Materials and methods for improving gastrointestinal function

ABSTRACT

The subject invention provides therapeutic compositions, and uses thereof for the treatment or amelioration of injury to small intestine mucosa. In preferred embodiments, the composition comprises one or more nutrients and/or electrolytes that acquire or retain considerable absorptive capacity.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation application of U.S. application Ser.No. 16/441,062, filed Jun. 14, 2019, which is a continuation applicationof U.S. application Ser. No. 14/656,255, filed Mar. 12, 2015, which is acontinuation application of U.S. application Ser. No. 13/245,430, filedSep. 26, 2011, which claims the priority benefit of U.S. ProvisionalApplication Ser. No. 61/386,317, filed Sep. 24, 2010, and U.S.Provisional Application Ser. No. 61/431,629, filed Jan. 11, 2011, all ofwhich are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.RC2-AI-087580 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in this invention.

BACKGROUND OF INVENTION

Radiation, a common therapy for malignancies in the abdomen and pelvis,can cause severe damage to the lining of the gastrointestinal (GI)tract, which is composed of rapidly dividing intestinal epithelialcells. Toxic effects of radiation on the gastrointestinal system causesymptoms such as nausea, vomiting, diarrhea, electrolyte imbalance anddehydration, and adversely affect patient recovery in the course ofcancer therapy. Even at low doses, a continuous loss of the villous andbrush border of the small bowel is observed within days afterirradiation. While crypt cells can rapidly repopulate the regionfollowing mild to moderate doses of (irradiation) IR, they became lostat a logarithmic rate after irradiation at high doses.

Irradiation is particularly destructive to the villous epithelium, wherenutrient and electrolyte absorption occurs. The villous epitheliumundergoes a continuous cellular loss and regeneration process, in whicha constant supply of immature enterocytes, originating from progenitorcells located within the lower poles of the crypts of Lieberkuhn,migrate out of the proliferative compartment at the base of the crypt tothe top of the villous. During their short lifespan, these enterocytesgradually mature along the crypt-villous axis into villous cells.Radiation therapy to the abdomen and pelvis region destroys not only theexisting villous cells, but also enterocytes from which new villouscells form, and thus, can deplete almost the entire villous epitheliumeven at moderate doses.

Due to the increasing use of high total radiation doses and cytotoxicagents, radiotherapy has been complicated by its acute GI toxicity.Damage to the GI tract not only results in malabsorption and loss ofnutrients and fluids, but also disrupts intestinal barrier function. Theleaky gut allows for easy entry of pathogens across the mucosal barrier,causing inflammation, bacteremia and endotoxemia. For instance, acuteradiation enteritis, diarrhea and abdominal pain can develop within dayspost irradiation even at doses as low as 5-12 Gy (a conventionalfractionated course of radiation uses 1.8-2 Gy per fraction), althoughGI toxicity usually occurs at higher doses. Chronic radiation enteritiscan develop between 18 months and 6 years after radiotherapy, while itmay develop even 15 years later²⁷⁻²⁹.

Treatment options for radiation enteritis are limited. Conventionaltreatment regimes include the administration of antidiarrheals toprevent fluid loss, smectite as an adsorbant of bile salts, opioids torelieve stomach or rectal pain, and steroids to relieve inflammation.Clinical trials have also investigated the efficacy of L. acidophilus,smectite or sucralfate for diarrhea prophylaxis, but only a moderatereduction of acute GI symptoms was achieved³⁰.

A common approach in the therapy of radiation enteritis is using totalparenteral nutrition (TPN) to provide intestinal rest. However, whetherparenteral nutrition satisfies the nutritional needs of patients, oractually has therapeutic effects on radiation enteritis remains to bedetermined. Although TPN may correct nutrition imbalance in certainpatients, severe radiation enteritis may still develop³⁷. TPN alsocauses intestinal atrophy, usually within 48 hours of administration.TPN also weakens mechanical and immunological barriers³⁸.

The exact biological mechanisms that lead to mucosal atrophy during TPN,which have not been well established, are believed to involve both localnutrient-sensing cell signals³⁹ and humoral signals, such as guthormones^(40,41). TPN has been shown to induce a rapid (<8 h) decreasein intestinal blood flow, which precedes villous atrophy and thesuppression of protein synthesis at 24 h, and cell proliferation andsurvival at 48 h⁴². In contrast, oral feeding rapidly increasesintestinal blood flow in neonatal and mature animals^(43,44). Similarly,in neonatal piglets, enteral feeding almost immediately (within 1-3hours) increases portal blood flow (PBF) up to 50% above values infood-deprived piglets⁴⁵. Thus, as shown in various studies, enteralfeeding is far superior to parenteral feeding^(7,8).

Currently, there is a lack of nutritional therapy that can effectivelyalleviate radiation enteritis. Although early studies suggested thatelemental or specific exclusion diets may be beneficial in selectedcases^(2,31,32), the efficacy of this approach has not been subsequentlyconfirmed. The current dietary therapy merely offers a means ofnutritional support to malnourished patients with chronic radiationenteritis.

Animal studies demonstrate that glutamine protects both upper and lowerGI tract mucosa from injury caused by chemotherapy or radiation therapy(RT)³³⁻³⁵. However, clinical trials fail to show that oral glutaminefeeding can prevent or alleviate acute diarrhea in patients who havereceived pelvic radiation therapy³⁶. Thus, a need exists for thedevelopment of improved feeding compositions for treatment ofirradiation-induced GI injury. As will be clear from the disclosuresthat follow, these and other benefits are provided by the subjectinvention.

BRIEF SUMMARY

The subject invention provides therapeutic compositions and methods forimproving small intestine function. The subject composition is usefulfor the treatment or amelioration of gastrointestinal injury associatedwith the loss of small intestine epithelial cells, particularly in thevillous region and the brush border, and/or for the treatment oramelioration of diseases or conditions associated with the alteration ofabsorptive capacity in the small intestine.

Advantageously, the subject therapeutic composition can be tailored tothe misbalanced absorptive state of the gastrointestinal system causedby the loss of small intestine epithelial cells and the alteration oftransport protein function in the small intestine. In a preferredembodiment, the subject composition is formulated for oraladministration.

In one embodiment, the therapeutic composition comprises, consistingessentially of, or consisting of, one or more free amino acids selectedfrom lysine, glycine, threonine, valine, tyrosine, aspartic acid,isoleucine, tryptophan, asparagine, and serine; and optionally,therapeutically acceptable carriers, electrolytes, vitamins, bufferingagents, and flavoring agents. The therapeutic composition isadministered via an enteral route. In one embodiment, the totalosmolarity of the composition is from about 230 mosm to 280 mosm, orpreferably, about 250 to 260 mosm. In one embodiment, the compositionhas a pH from about 7.1 to 7.9, preferably, about 7.4.

In a specific embodiment, the composition of the subject invention doesnot comprise glucose, glutamine, methionine, and/or lactose.

Also provided are methods for treatment or amelioration of diseases orsymptoms associated with the loss of small intestine epithelial cells,particularly in the villous region and brush border, and diseases orsymptoms associated with the alteration of transport protein function inthe small intestine epithelium. The method comprises administering, viaan enteral route, to a subject in need of such treatment, an effectiveamount of the composition of the subject invention. Preferably, thesubject composition is administered orally and reaches the intestine ofthe subject.

The subject invention also provides methods for preparing thetherapeutic composition, and for screening for nutrients or electrolytesfor inclusion into the subject therapeutic/dietary composition, byselecting nutrients or electrolytes that retain or acquire considerableabsorptive capacity following the destruction of small intestineepithelial cells. These methods can be adapted for use in individualpatients, thereby facilitating the development of compositions andmethods specifically designed to meet the needs of an individualpatient.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B show effect of irradiation (IR) on net anion secretion (1A)and conductance (1B). (1A). 12 Gy IR tissues were studied on day 1, 3and 4. Maximal increase in I_(sc) was seen on day 2. Arrow representsthe time point when forskolin was added. (1B). Effect of increased dosesof IR on net anion secretion. All the tissues were studied on day 6 andn=12. The results showed an IR dose-dependent increase in conductance.

FIGS. 2A-2D show change in I_(sc) with increasing dose of irradiation.All the values are derived from n=24 tissues. Experiments were performedon day 4 post-irradiation in regular Ringer solution on both sides ofthe chamber with a total osmolarity of 296 mosm. Histopathology sectionsshowed minimal villous and crypt damage at 3 Gy, and extensive villousand crypt damage at 7 Gy as compared to 0 Gy.

FIG. 3A shows change in I_(sc) in mice epithelial cells over timefollowing irradiation at 3 Gy. Values represent mean S.E.M. n=6 tissues.Maximal increase in I_(sc) was seen on 6th day following irradiation. Nosignificant difference was seen between 5th, 6th and 7th days. Withtime >7 days post-irradiation, there was a slight decrease in I_(sc) ascompared to that of day 5, 6 or 7. I_(sc) values of day 5, 6, and 7 weresimilar.

FIG. 3B shows ion transport of a small intestine epithelial cell.

FIG. 3C shows the effect of bumetanide on basal and cAMP-stimulatedI_(sc) in non-irradiated and 3-Gy irradiated tissues.

FIG. 3D shows the contribution of HCO₃ ⁻ in net anion secretion. Thiswas determined by replacing Cl⁻ in Ringer solution with equimolaramounts of isethionate. Forskolin stimulated an increase in I_(sc) in 0Gy (*. p<0.02) but not in 3 Gy tissues.

FIG. 3E shows effect of bath Na⁺ on HCO₃ ⁻ secretion. All of the resultsshown in FIG. 3 are from n=6 tissues. Error bars represent SEM.

FIG. 4A shows changes in plasma endotoxin level following IR. Plasmaendotoxin levels were measured on day 6, post-IR.

FIG. 4B shows changes in permeability ratio of Cl⁻ & Na⁺ plotted againstchanges in membrane voltage (Dilution potential). Irradiation at 7 Gycauses a complete loss of selectivity.

FIGS. 5A-5C show that irradiation increases levels of inflammatorymediators, including IL-1β, TNFα and MIP-{tilde over (α)}

FIGS. 6A-6E show changes in HCO₃ ⁻ secretion due to irradiation andimmunostaining for HCO₃ ⁻ secretory machinery. (6A) shows effects ofirradiation on bath Na⁺ on HCO₃ ⁻ secretion. Experiments were performedin A) in Cl-containing solutions with 140 mM Na⁺ or B) Cl⁻ containingsolutions without Na⁺. Tissues were stimulated with forskolin. HCO₃ ⁻secretion was compared to that of between 0 Gy and 3Gy irradiated mice.Significantly higher bath Na⁺-dependent HCO₃ ⁻ secretion was observed in0 Gy as compared to 3 Gy irradiated mice (p<0.001). Results are derivedfrom n=6 tissues. Error bars represent S.E.M. (6B-6E) showimmunostaining of jejunum tissues of mice received 0 Gy and 3 Gyirradiation, using NBCe1 a/b antibody.

FIGS. 7A-7B show IR dose-dependent changes in glucose transport andkinetics. (7A) shows that irradiation resulted in a dose-dependentdecrease in glucose-stimulated Na⁺ I_(sc) measured in Ussing chamber.(7B) shows decreased SGLT1 affinity for glucose as irradiation dosesincreased.

FIG. 8 shows that irradiation reduced glucose-stimulated current in adose-dependent manner starting from irradiation at 1 Gy. Irradiation at7 Gy almost completely inactivated glucose transport.

FIG. 9A displays short-circuit current, showing saturated kinetics withincrease in glucose concentration. Particularly, glucose transport issaturated at a concentration of 4 mM.

FIG. 9B shows irradiation dose-dependent increase in K_(m) values. Themaximal increase in K_(m) was observed at 7 Gy. This indicates thatirradiation caused decreased affinity of SGLT-1 to glucose.

FIG. 10 shows that V_(max) decreased as irradiation doses increased. Theminimal decrease in V_(max) was observed at 7 Gy. This indicates thatirradiation causes a reduction of functional SGLT-1 for glucosetransport.

FIG. 11 shows changes in K_(m) over time post irradiation. K_(m)increased immediately after irradiation and returned to normal (controlvalues) approximately 14 days post irradiation.

FIGS. 12A-12B show results of murine survival studies after 9-Gy and15.6-Gy irradiation. Death of glucose-treated mice occur starting ondays 5 and 7, while control mice did not die until 10 days afterirradiation. On day 20, 30% of the control mice were alive, whereas noneof the glucose-treated mice survived on day 20.

FIG. 13 shows Western blot analysis of SGLT-1 protein levels inwhole-cell lysates. The results showed that irradiation increased SGLT-1expression.

FIG. 14 shows Western blot analysis of SGLT-1 protein levels inbrush-border membrane vesicles of jejunum tissues. Irradiation increasedSGLT-1 protein levels in a dose-dependent manner. No SGLT-1 protein wasdetected in colonic tissues.

FIG. 15 shows that irradiation caused a dose-dependent increase inglutamine-stimulated I_(sc).

FIG. 16 shows that irradiation caused a dose-dependent decrease inlysine-stimulated I_(sc).

FIGS. 17A-17B show mice survival rate following lysine (17A) or glucose(17B) therapy after IR. Lysine administration resulted in increasedsurvival, whereas glucose administration resulted in decreased survival.

FIGS. 18A-18D show Western blot analyses for various transport proteins.Western blot analysis showing NKCC1 (18A), CFTR (18C) and NBCe1-A/B(18B) protein levels in jejunum of mice. From left to right, the lanesrepresent 0, 1, 3, 5 and 7 Gy. Irradiation increased NKCC1 proteinlevels from 1-5 Gy and such increase decreased at 7 Gy (18A). NBCe1-A/Bprotein levels significantly decreased following irradiation. CFTR (18C)protein levels in jejunum tissues significantly increased followingirradiation at 3 Gy as compared to 0 Gy. Jejunum had the highestNBCe1-A/B protein levels compared to that in duodenum, ileum or colon(18D). Tissues were harvested for western blot on day 6post-irradiation.

FIGS. 19A-19B show schematic models for cAMP-stimulated (19A) andirradiation-induced (19B) anion secretion.

FIGS. 20A-20B show injury to small intestine mucosa in mice treated with5-fluorouracil (5-FU) (20A) and cisplatin (20B). (20A) shows change inI_(SC) in 5-FU-injected mice. (20B) shows change in I_(SC) incisplatin-injected mice.

FIGS. 21A-21B show that the administration of with the therapeuticcomposition of the subject invention improves small intestine functionof mice that have received 5-U.

DETAILED DISCLOSURE

The subject invention provides therapeutic compositions and methods forimproving small intestine function. The composition is formulated forenteral administration. The compositions and methods of the subjectinvention are particularly useful for the treatment or amelioration ofgastrointestinal injury associated with the loss of small intestineepithelial cells, particularly in the villous region and brush border,and/or for the treatment of diseases or conditions associated with thealteration of transport protein function in the small intestineepithelium.

Advantageously, the subject therapeutic composition is tailored to themisbalanced absorptive state of the gastrointestinal system caused bythe loss of small intestine epithelial cells, particularly, in the smallintestine villous region and brush border, as well as the alteration oftransport protein function. Particularly, the subject invention canimprove small intestine mucosal healing, restore small intestinefunction, enhance fluid retention, prevent or alleviate small intestineatrophy, and/or restore or enhance small intestine barrier function of apatient having injury to the small intestine mucosa.

In one embodiment, the therapeutic composition comprises, consistsessentially of, or consists of one or more free amino acids selectedfrom lysine, glycine, threonine, valine, tyrosine, aspartic acid,isoleucine, tryptophan, asparagine, and serine; and optionally,therapeutically acceptable carriers, electrolytes, vitamins, bufferingagents, and flavoring agents. The therapeutic composition isadministered via an enteral route. In one embodiment, the totalosmolarity of the composition is from about 230 mosm to 280 mosm, orpreferably, is about 250 to 260 mosm. In one embodiment, the compositionhas a pH from about 4.0 to 8.5, preferably 5.0 to 8.2, more preferably6.0 to 8.0, more preferably, 7.1 to 7.9, and most preferably, about 7.4.

In a specific embodiment, the composition of the subject invention doesnot comprise glucose, glutamine, methionine, and/or lactose.

Also provided are methods for the treatment or amelioration of diseasesor conditions associated with the loss of small intestine epithelialcells, particularly in the villous region and brush border, and diseasesor conditions associated with the alteration of transport proteinfunction in the small intestine epithelium. The method comprisesadministering via an enteral route, to a subject in need of suchtreatment, an effective amount of the composition of the subjectinvention.

The subject invention is based, at least in part, on the discovery thatenteral feeding to subjects with only the nutrients that retain oracquire sufficient absorptive capacity following injury to the smallintestine mucosa improves mucosal healing, restores small intestinefunction, enhances fluid retention, and alleviates an array ofassociated disease symptoms including, but not limited to,malabsorption, diarrhea, nausea, vomiting, electrolyte imbalance, anddehydration.

In accordance with the subject invention, it has been determined that,following radiation and chemotherapy, an alteration in transport proteinfunction is observed with respect to, for example, glucose, glutamine,and lysine, and electrolytes such as Na⁺, HCO₃ ⁻, and Cl⁻. In addition,radiation causes increased net anion secretion. The alterations ofnutrient and electrolyte absorptive capacity occur immediately afterradiation and chemotherapy, but it is possible for the absorptivecapacity to recover towards normal (about 8-14 days post-irradiation inmice models).

Specifically, radiation causes an irradiation dose-dependent decrease inglucose absorption due to the reduced affinity of the sodium-dependentglucose transport system (SGLT-1) to glucose. Functional studies onglucose-stimulation showed that radiation caused a dose-dependentdecrease in glucose-transport activity and decreased affinity of SGLT-1for glucose.

It is known that the presence of unabsorbed nutrients or solutes in theintestinal lumen can lead to osmotic diarrhea. In accordance with thesubject invention, oral feeding of an irradiated subject with glucoseand/or glutamine has been found to cause osmotic diarrhea and reducedsurvival, while oral feeding of each, or a combination of, amino acidsselected from lysine, glycine, threonine, valine, tyrosine, asparticacid, isoleucine, tryptophan, asparagine, and/or serine, prolongssurvival.

Therapeutic Composition for Improving Small Intestine Function

In one aspect, the subject invention provides therapeutic compositionsfor improving small intestine function. In one embodiment, thetherapeutic composition comprises, consisting essentially of, orconsisting of, one or more free amino acids selected from lysine,glycine, threonine, valine, tyrosine, aspartic acid, isoleucine,tryptophan, asparagine, and serine; and optionally, therapeuticallyacceptable carriers, electrolytes, vitamins, buffering agents, andflavoring agents. The therapeutic composition is administered via anenteral route.

Preferably, the composition is slightly alkaline and is hypotonic whencompared to the osmotic pressure of small intestine epithelial cells(such as villous cells and crypt cells of the small intestine).Preferably, the subject composition comprises water. Preferably, thecomposition is formulated as an oral rehydration drink for improvingsmall intestine function that is undermined due to the loss of, orinjury to, villous epithelial cells.

In one embodiment, the total osmolarity of the composition is from about230 mosm to 280 mosm, or any value therebetween. Preferably, the totalosmolarity is from about 250 to 260 mosm. In another embodiment, thecomposition has a total osmolarity that is any value lower than 280mosm.

In one embodiment, the composition has a pH from about 7.1 to 7.9, orany value therebetween. Preferably, the composition has a pH from about7.3 to 7.5, more preferably, about 7.4.

In certain embodiments, each free amino acid can be present at aconcentration from 4 mM to 40 mM, or any value therebetween, wherein thetotal osmolarity of the composition is from about 230 mosm to 280 mosm.Alternatively, if the amino acid concentration is calculated based onmg/l, each free amino acid can be present at a concentration from 300mg/l to 8000 mg/L, or any value therebetween, wherein the totalosmolarity of the composition is from about 240 mosm to 280 mosm.

In certain specific embodiments, the therapeutic composition comprisesone or more free amino acids present at their respective concentrationsas follows: lysine at a concentration of about 730 to 6575 mg/l, or anyvalue therebetween; aspartic acid at a concentration of about 532 to4792 mg/l, or any value therebetween; glycine at a concentration ofabout 300 to 2703 mg/l, or any value therebetween; isoleucine at aconcentration of about 525 to 4722 mg/l, or any value therebetween;threonine at a concentration of about 476 to 4288 mg/l, or any valuetherebetween; tyrosine at a concentration of about 725 to 6523 mg/l, orany value therebetween; valine at a concentration of about 469 to 4217mg/l, or any value therebetween; tryptophan at a concentration of about817 to 7352 mg/i, or any value therebetween; asparagine at aconcentration of about 528 to 4756 mg/l, or any value therebetween;and/or serine at a concentration of about 420 to 3784 mg/l, or any valuetherebetween; wherein the total osmolarity of the composition is fromabout 240 mosm to 280 mosm.

In one embodiment, the subject invention provides a drink comprising thefollowing constituents lysine (11-21 mosm), aspartic acid (3-13 mosm),glycine (19-29 mosm), isoleucine (19-29 mosm), threonine (19-29 mosm),tyrosine (0.5-5 mosm), valine (19-29 mosm), tryptophan (5-20 mosm),asparagine (3-13 mosm), and serine (3-8 mosm), or a subset of theseingredients.

In one specific embodiment, the composition comprises lysine, glycine,threonine, valine, and tyrosine in a form of free amino acids. In afurther specific embodiment, the composition comprises lysine, glycine,threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan,asparagine, and serine in a form of free amino acids.

In a further embodiment, the composition comprises one or moredipeptides that are made of the same or different amino acids selectedfrom lysine, glycine, threonine, valine, tyrosine, aspartic acid,isoleucine, tryptophan, asparagine, or serine.

In one embodiment, the composition does not contain glutamine and/ormethionine; and any di-, oligo-, or polypeptides or proteins that can behydrolyzed into glutamine and/or methionine.

In an alternative embodiment, the composition may comprise free aminoacid glutamine, and, optionally, one or more glutamine-containingdipeptides, wherein the total concentration of the free amino acidglutamine and the glutamine-containing dipeptide(s) is less than 300mg/l, or any concentrations lower than 300 mg/l, such as 100 mg/l, 50mg/l, 10 mg/l, 5 mg/l, 1 mg/l, 0.5 mg/l, or 0.01 mg/l.

In another alternative embodiment, the therapeutic composition maycomprise free amino acid methionine, and, optionally, one or moremethionine-containing dipeptides, wherein the total concentration of thefree amino acid methionine and the methionine-containing dipeptide(s) isless than 300 mg/l, or any concentrations lower than 300 mg/l, such as100 mg/l, 50 mg/l, 10 mg/l, 5 mg/l, 1 mg/l, 0.5 mg/l, or 0.01 mg/l.

In one embodiment, the therapeutic composition does not contain anysaccharides, including any mono-, di-, oligo-, polysaccharides, andcarbohydrates. In one specific embodiment, the therapeutic compositiondoes not contain glucose, and/or any di-, oligo, polysaccharides, andcarbohydrates that can be hydrolyzed into glucose. In a specificembodiment, the composition does not contain lactose. In anotherspecific embodiment, the therapeutic composition does not containfructose and/or galactose, and/or any di-, oligo, polysaccharides, andcarbohydrates that can be hydrolyzed into fructose and/or galactose.

In an alternative embodiment, the therapeutic composition may comprisemonosaccharide glucose, and, optionally, one or more glucose-containingdisaccharides other than lactose, wherein the total concentration of themonosaccharide glucose and the glucose-containing disaccharide(s) isless than 3 g/l, or any concentrations lower than 3 g/l, such as 1 g/l,500 mg/l, 300 mg/l, 100 mg/l, 50 mg/l, 10 mg/l, 5 mg/l, 1 mg/l, 0.5mg/l, or 0.01 mg/l.

In certain embodiments, the therapeutic composition comprises one ormore electrolytes selected from, for example, Na⁺; K⁺; HCO₃ ⁻; C₃ ²⁻;Ca²⁺; Mg²⁺; Fe²; Cl⁻; phosphate ions, such as H₂PO₄ ⁻, HPO₄ ²⁻, and PO₄³⁻; zinc; iodine; copper; iron; selenium; chromium; and molybdenum. Inan alternative embodiment, the composition does not contain HCO₃ ⁻ orCO₃ ²⁻. In another alternative embodiment, the composition comprisesHCO₃ ⁻ and CO₃ ²⁻ at a total concentration of less than 5 mg/l, orconcentrations lower than 5 mg/l.

In a further embodiment, the therapeutic composition comprises one ormore vitamins including, but not limited to, vitamin A, vitamin C,vitamin D (e.g., vitamin D₁, D₂, D₃, D₄, and/or D₅), vitamin E, vitaminB₁ (thiamine), vitamin B₂ (e.g., riboflavin), vitamin B₃ (e.g., niacinor niacinamide), vitamin B₅ (pantothenic acid), vitamin B₆ (pyridoxine),vitamin B₇ (biotin), vitamin B₉ (e.g., folate or folic acid), vitaminB₁₂ (cobalamin), and vitamin K (e.g., vitamin K₁, K₂, K₃, K₄, and K₅),and choline.

In certain embodiments, the composition does not contain one or more ofthe ingredients selected from oligo-, polysaccharides, andcarbohydrates; oligo-, or polypeptides or proteins; lipids; small-,medium-, and/or long-chain fatty acids; and/or food containing one ormore above-mentioned nutrients.

In one embodiment, phosphate ions, such as H₂PO₄ ⁻, HPO₄ ²⁻, and PO₄ ³⁻,are used to buffer the composition of the subject invention. In oneembodiment, the therapeutic composition uses HCO₃ ⁻ or CO₃ ²⁻ as abuffer. In another embodiment, the therapeutic composition does not useHCO₃ ⁻ or CO₃ ²⁻ as buffer.

The term “consisting essentially of,” as used herein, limits the scopeof the ingredients and steps to the specified materials or steps andthose that do not materially affect the basic and novelcharacteristic(s) of the present invention, i.e., compositions andmethods for treatment of injury to small intestine epithelium,particularly in the villous region and brush border. For instance, byusing “consisting essentially of,” the therapeutic composition does notcontain any unspecified ingredients including, but not limited to, freeamino acids, di-, oligo-, or polypeptides or proteins; and mono-, di-,oligo-, polysaccharides, and carbohydrates that have a direct beneficialor adverse therapeutic effect on treatment of injury to small intestineepithelium, particularly in the villous region and brush border. Also,by using the term “consisting essentially of,” the compositing maycomprise substances that do not have therapeutic effects on thetreatment of injury to small intestine epithelium; such ingredientsinclude carriers, excipients, adjuvants, flavoring agents, etc that donot affect the health or function of the injured small intestineepithelium, particularly in the villous region and brush border.

The term “oligopeptide,” as used herein, refers to a peptide consistingof three to twenty amino acids. The term “oligosaccharides,” as usedherein, refers to a saccharide consisting of three to twentymonosaccharides.

In one embodiment, the composition of the subject invention comprisesnutrients (such as free amino acids) and/or electrolytes that retain oracquire improved absorptive capacity in a subject having injury to smallintestine epithelial cells, when compared to the absorptive capacity ofnormal controls who do not have injury to small intestine epithelialcells (such as villous cells, crypt cells, enterocytes, and intestinalprojenitor cells).

In a further embodiment, the composition of the subject invention doesnot contain nutrients (such as amino acids) and/or electrolytes that arenot absorbed, or have reduced absorption, in a subject having injury tosmall intestine epithelial cells, when compared to the absorptivecapacity of normal controls who do not have injury to small intestineepithelial cells (such as villous cells, crypt cells, enterocytes, andintestinal projenitor cells). Advantageously, the compositions of thesubject invention facilitate easy absorption of nutrients by theintestine to reduce undue energy expenditure, thereby providingintestinal rest in the immediate time period after mucosal injury.

Treatment Method for Improving Small Intestine Function

Another aspect of the subject invention provides methods for treatmentor amelioration of diseases or conditions associated with the loss of,or injury to, small intestine epithelial cells, particularly in thevillous region and brush border. In one embodiment, the loss of, orinjury to, small intestine epithelial cells results in alteredabsorptive capacity for nutrients, electrolytes, and/or fluids.Advantageously, to patients with the loss of, or injury to, smallintestine epithelial cells, particularly to patients with smallintestine villous atrophy, the subject invention improves smallintestine mucosal healing; improves small intestine function; enhancesabsorption of nutrients and fluid retention in the small intestine;prevents or alleviates small intestine atrophy; alleviates abdominalpain; prevents and/or treats diarrhea; restores or enhances smallintestine barrier function; and/or reduces small intestine mucosalinflammation, bacteremia and/or endotoxemia.

Accordingly, the subject invention is particularly beneficial forimproving gastrointestinal health of subjects that receive cytotoxicchemotherapeutic agents, pelvic or abdominal radiation, proton therapy,and abdominal surgery; subjects that suffer from infection or autoimmunediseases associated with acute or chronic inflammation in the smallintestine; subjects that are routinely, or accidentally exposed toradiation, such as for example, astronauts and pilots who are routinelyexposed to space radiation, and subjects exposed to radiation due tonuclear accident, acts of war, or terrorism.

In one embodiment, the method comprises administering, via an enteralroute, to a patient or subject in need of such treatment, an effectiveamount of a composition of the invention. The composition can beadministered to a patient or subject immediately before, during, and/orafter injury to small intestine epithelial cells, and can beadministered once or multiple times each day.

The term “subject” or “patient,” as used herein, describes an organism,including mammals such as primates, to which treatment with thecompositions according to the present invention can be provided.Mammalian species that can benefit from the disclosed methods oftreatment include, but are not limited to, apes, chimpanzees,orangutans, humans, monkeys; domesticated animals such as dogs, cats;live stocks such as horses, cattle, pigs, sheep, goats, chickens; andanimals such as mice, rats, guinea pigs, and hamsters.

In one specific embodiment, a subject in need of treatment is a patientwith injury to small intestine mucosal epithelial cells, including themucosa layer of duodenum, jejunum, and ileum. Particularly, a subject inneed of treatment is a patient with injury to the villous region andbrush border of the small intestine. For instance, the subject in needof treatment has villous atrophy (e.g., partial or complete wasting awayof the villous region and brush border); has at least a 5% (such as atleast 10%, 20%, 30%, or 50%) reduction in villous cells in the smallintestine; has lost at least 5% (such as at least 10%, 20%, 30%, or 50%)villous height when compared to normal; has a loss of function of one ormore transporters in the villous region and brush border of the smallintestine, wherein the transporters include, but are not limited to, theSGLT-1 transporter, the AE2 transporter, the NHE1 transporter, and theNBCe1-A/B transporter, wherein the loss of transporter function is atleast 5% (such as at least 10%, 20%, 30%, or 50%); and/or has a changein absorptive capacity of one or more nutrients in the small intestine,wherein the nutrients are selected from isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan, valine, histidine,tyrosine, alanine, arginine, glutamine, aspartic acid, aspartate,cysteine, glycine, proline, serine, asparagine, glucose, fructose,and/or lactose, wherein the change in absorptive capacity is at least 5%(such as at least 10%, 20%, 30%, or 50%).

Changes in absorptive capacity of the small bowel can be determined by,for example, using an Ussing Chamber, as illustrated in the Materialsand Methods section herein. For example, the changes in absorptive statecan be determined by, for example, measuring a combination of indicesincluding, for example, K_(m), V_(max), and I_(sc). Injury to thevillous and other regions of the small intestine can be determined by,for example, examination of biopsy samples of small intestine mucosa.

Diseases and therapeutic procedures that cause injury to small intestinemucosal epithelial cells, such as small intestine villous cells, can bereadily determined by a skilled clinician. As is known in the medicalprofession, patients with certain diseases, such as inflammator boweldisease (IBD), ulcerative colitis, duodenal ulcers, and Crohn's disease,suffer from chronic destruction of the small intestine mucosa.Radiation, chemo-, and proton therapy also cause injury to smallintestine cells.

The term “treatment” or any grammatical variation thereof (e.g., treat,treating, and treatment etc.), as used herein, includes but is notlimited to, alleviating a symptom of a disease or condition; and/orreducing, suppressing, inhibiting, lessening, or affecting theprogression, severity, and/or scope of a disease or condition.

The term “amelioration” or any grammatical variation thereof (e.g.,ameliorate, ameliorating, and amelioration etc.), as used herein,includes, but is not limited to, delaying the onset, or reducing theseverity of a disease or condition (e.g., diarrhea, bacteremia and/orendotoxemia). Amelioration, as used herein, does not require thecomplete absence of symptoms.

The term “effective amount,” as used herein, refers to an amount that iscapable of treating or ameliorating a disease or condition or otherwisecapable of producing an intended therapeutic effect.

In one specific embodiment, the subject invention provides a method forpromoting intestinal health of a subject with injury to small intestineepithelial cells, wherein said method comprises: identifying a subjectwith injury to small intestine epithelial cells, or who is about to beinflicted with such an injury, and is in need of treatment oramelioration, and administering, via an enteral route, to the subject,an effective amount of a composition comprising, consisting essentiallyof, or consisting of one or more free amino acids selected from lysine,glycine, threonine, valine, tyrosine, aspartic acid, isoleucine,tryptophan, asparagine, and serine; water; and optionally,therapeutically acceptable carriers, electrolytes, vitamins, bufferingagents, and flavoring agents, wherein the composition has a totalosmolarity from 240 to 280 mosm and a pH of about 7.1 to 7.9.

In one embodiment, one or more of the following nutrients are notadministered, via an enteral route, to a subject with (or about to have)injury to small intestine epithelial cells, wherein the nutrients areselected from glutamine, methionine, and any di-, oligo-, orpolypeptides or proteins that can be hydrolyzed into glutamine and/ormethionine; glucose and any di-, oligo, polysaccharides, andcarbohydrates that can be hydrolyzed to glucose; and/or food that, upondigestion, requires absorption of any of the above-mentioned nutrientsin the small intestine.

In a further embodiment, for a subject with (or about to have) injury tosmall intestine epithelial cells, none of the following nutrients areadministered via an enteral route, wherein the nutrients are selectedfrom saccharides, lipids, fatty acids, and/or food that, upon digestion,requires absorption of any of the above-mentioned nutrients in the smallintestine. For patients that are exposed to radiation, or receiveradiation, chemo-, and proton therapy, injury to small intestineepithelial cells typically lasts for at least 3, 7, 14, 21, 30 days, orany period between 1-30 days.

In a further embodiment, after any period between 1-30 days (such asafter 3, 7, 14, 21, 30 days) since the subject is exposed to radiation,or receives radiation, chemo-, and/or proton therapy, one or more of thefollowing nutrients are administered via an enteral route for enhancingmucosal healing, wherein the nutrients are selected from: glutamine,methionine, and any di-, oligo-, or polypeptides or proteins that can behydrolyzed into glutamine and/or methionine; glucose and any di-, oligo,polysaccharides, and carbohydrates that can be hydrolyzed to glucose;and/or food that, upon digestion, requires absorption of any of theabove-mentioned nutrients in the small intestine.

In a specific embodiment, the subject composition is administered orallyand reaches the small intestine of the subject. Optionally, the methodfurther comprises administering, via a parenteral route, requirednutrients and electrolytes that are not administered in sufficientamounts via the enteral route.

In one embodiment, the subject invention is not used to providesignificant amounts or all of the essential nutrition to a subject, butis to improve small intestine mucosal healing, restore small intestinefunction, enhance fluid retention, prevent or alleviate small intestinevillous atrophy, prevent and/or treat diarrhea, and/or restore orenhance intestinal barrier function. In a specific embodiment, thecomposition of the drink is also based on improvement in the barrierfunction. Barrier function can be determined using multiple techniquesincluding: a) an increase in conductance measurements on tissues mountedin a Ussing chamber, b) dilution potential used to measure relativepermeability of Cl and Na (PCl/PNa) (only intact and functional barriercan maintain ion selectivity; when the barrier function is lost, the ionselective ratio is close to one), and c) measuring plasma endotoxinlevels. When mucosal barrier function is lost the commensal gut bacteriacan find their way into the systemic circulation, resulting is raisedplasma endotoxin levels. Endotoxin levels can be measured in a patient'sblood sample. Plasma endotoxin levels can also be used as an index tomeasure improvement with treatment.

The compositions of the subject invention can be used in the treatmentor amelioration of any diseases or conditions associated with the loss,destruction, or reduction of small intestine epithelial cells,particularly the loss, destruction, or reduction in function or numberof villous cells, enterocytes, and/or intestinal progenitor cells of thesmall intestine. The subject invention is particularly useful for thetreatment or amelioration of any diseases or conditions associated withthe loss, inactivation, or functional alteration of transport proteinsin the small intestine epithelial cells, particularly transport proteinsin the villous cells of the small intestine.

In one embodiment, the compositions and methods of the subject inventioncan be used in the treatment or amelioration of a disease or conditionarising from, or associated with, a reduced affinity of sodium-dependentglucose transport system (SGLT-1) to glucose; a loss or reduced activityof NH₂-terminal electrogenic Na+-HCO₃(−) cotransporter (NBCe1-A/B); aloss or reduced activity of apical Cl⁻—HCO₃ ⁻ exchange transporter(AE1); and/or an increased level or activity of CFTR and/or NKCC-1transporter systems.

In a specifically preferred embodiment, the compositions and methods ofthe subject invention can be used in the treatment or amelioration ofinjury to the small intestine caused by radiation. In a specificembodiment, the subject invention can be used in the treatment oramelioration of injury to the small intestine caused by radiationtherapy, particularly pelvic and abdominal radiation therapy. In aspecific embodiment, the radiation therapy is for cancer treatment.

In addition, the subject invention can be used in the treatment oramelioration of injury to the small intestine caused by routineradiation exposure, such as exposure to space radiation in astronautsand pilots; radiation exposure, such as by a radioactive weapon andaccidental nuclear release. Specifically, the subject invention can beused to treat or ameliorate acute and/or chronic radiation enteritis.

In certain specific embodiments, the compositions and methods of thesubject invention can be used in the treatment or amelioration of injuryto the small intestine, wherein the patient received radiation at 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 Gy.In another embodiment, the subject received radiation at a dose higherthan 20 Gy.

Additionally, the subject invention can be used in the treatment oramelioration of injury to the small intestine caused by chemotherapeuticagents including, but not limited to, cisplatin, 5-fluorouracil (5-FU),hydroxyurea, etoposide, arabinoside, 6-mercaptopurine, 6-thioguanine,fludarabine, methothexate, steroids, and/or a combination thereof.

In addition, the subject invention can be used in the treatment oramelioration of injury to the small intestine caused by proton therapy.

In certain embodiments, the subject invention can be used in thetreatment or amelioration of diseases involving injury to the smallintestine including, but not limited to, inflammatory bowel disease(IBD), ulcerative colitis, duodenal ulcers, Crohn's disease, and/orcoeliac disease (also known as celiac disease). The subject inventioncan be used in the treatment or amelioration of injury to the smallintestine due to pathogenic infection, such as viral, bacterial, fungalor other microbial infection.

In one specific embodiment, the subject invention can be used in thetreatment or amelioration of small intestine villous atrophy, i.e.,partial or complete wasting away of the villous region and brush border,as well as diseases and conditions that arise from, associated with,and/or are caused by small intestinal villous atrophy.

In certain embodiments, the subject invention can be used in thetreatment or amelioration of focal villous atrophy and/or diffusevillous atrophy; hyperplastic villous atrophy and/or hypoplastic villousatrophy; and/or villous atrophy with and without mucosal inflammation.

In certain embodiments, the subject invention can be used in thetreatment or amelioration of hyperplastic villous atrophy (with crypthyperplasia) and associated diseases and conditions including, but notlimited to, coeliac disease (with gluten-sensitive enteropathy); chronictrauma; small bowell transplantion; urinary ileal conduits; intestinalmucosal inflammation; intestinal ulcers; intestinal anastomosis;glucagonoma; extensive small bowel resections; primary ileal villousatrophy; microscopic colitis atrophy; intestinal microvillous atrophy;and mitochondrial cytopathy (mitochondrial respiratory chain anomaly).

In certain embodiments, the subject invention can be used in thetreatment or amelioration of hypoplastic villous atrophy (without crypthyperplasia) and associated diseases and conditions including, but notlimited to, malignancy; paneth cell deficiency; hypopituitarism; coeliacdisease unresponsive to gluten-free diet; tropical sprue;radiation-associated ischemia; drug-induced villous atrophy, such asvillous atrophy induced by neomycin and azathioprin.

In certain embodiments, the subject invention can be used in thetreatment or amelioration of villous atrophy with mucosal inflammationas well as associated diseases and conditions including, but not limitedto, coeliac disease; severe alimentary intolerance; congenital Crohndisease; autoimmune enteropathy; enterocolitis; and immunodeficiencysyndromes.

In certain embodiments, the subject invention can be used in thetreatment or amelioration of villous atrophy that are caused by diseasesincluding, but not limited to, hepatitis; intestinal cancer; intestinallymphoma; type 1 diabetes; allergy; eosinophillic gastroenteritis; viralgastroenteritis; and autoimmune enteropathy.

In certain embodiments, the subject invention can be used in thetreatment or amelioration of villous atrophy associated with coeliacdisease in the small bowel, including but not limited to, Marsh type 3avillous atrophy (>40 intraepithelial lymphocytes per 100 enterocytes;mild villous atrophy), Marsh type 3b villous atrophy (>40intraepithelial lymphocytes per 100 enterocytes; marked villousatrophy), Marsh type 3c villous atrophy (>40 intraepithelial lymphocytesper 100 enterocytes; villous region absent o almost absent), (based onmodified Marsh classification of coeliac disease and intestinal villousatrophy).

The subject invention can also be used to treat or ameliorate symptomsassociated with injury to the small intestine including, but not limitedto, malabsorption, diarrhea, nausea, vomiting, electrolyte imbalance,malabsorption, and dehydration.

Preparation of Therapeutic Composition for Improving Small IntestineFunction

In another aspect, a method for preparing the therapeutic composition ofthe invention is provided. In one embodiment, the method comprisespreparing a composition for promoting intestinal health of a subjectwith the loss of, or injury to, small intestine epithelial cells,wherein the composition comprises, consists essentially of, or consistsof an effective amount of one or more ingredients, wherein theingredients are absorbed by the small intestine of a subject with a lossof, or injury to, small intestine epithelial cells, wherein thecomposition has a total osmolarity from 230 mosm to 280 mosm, or anyvalue therebetween (preferably about 250 mosm to 260 mosm), wherein thecomposition has a pH of about 7.1 to 7.9, or any value therebetween(preferably about 7.4), and wherein the composition is formulated forenteral administration.

In one embodiment, the ingredients are selected from free amino acids,dipeptides, monosaccharides, disaccharides, or a combination thereof,and, optionally, electrolytes, vitamins, flavoring agents, and/orcarriers.

In one embodiment, the subject invention provides methods for screeningfor nutrients or electrolytes for inclusion into the subject therapeuticcomposition, by selecting nutrients or electrolytes that retain oracquire absorptive capacity following the destruction of small intestineepithelial cells in the villous and crypt regions.

The subject screening methods can be used for determining therapeuticnutrients and/or electrolytes that can be used in the treatment oramelioration of diseases or conditions associated with the loss,destruction, or reduction of small intestine epithelial cells,particularly the loss, destruction, or reduction of villous cells,enterocytes, and/or intestinal progenitor cells. In specificembodiments, the methods can be used to design compositions and methodsto meet the needs of a specific patient or group of patients. In aspecific embodiment, the subject composition is useful for the treatmentor amelioration of injury to small intestine following radiation,chemo-, proton therapy, or due to acute or chronic inflammation in thesmall intestine.

In one embodiment, the subject screening method comprises:

a) contacting small intestine epithelial tissue having injury in themucosa with a candidate nutrient or electrolyte;

b) determining a level of the ability of the small intestine epithelialtissue for absorbing said nutrient or electrolyte;

c) comparing said level to a predetermined level (such as in normaltissues); and

d) selecting the candidate nutrient or electrolyte if the absorptiveability of the candidate nutrient or electrolyte is at least, forexample, 50%, 60%, 70%, 80%, or 90% of the predetermined level.

In one embodiment, the subject screening method comprises:

a) administering, via an enteral route, a candidate nutrient orelectrolyte to a subject with injury to the small intestine mocusa;

b) determining a level of intestinal absorptive capacity of saidnutrient or electrolyte;

c) comparing said level to a predetermined level (such as in normalsubjects); and

d) selecting the candidate nutrient or electrolyte if the absorptivelevel of the candidate nutrient or electrolyte is at least, for example,50%, 60%, 70%, 80%, or 90% of the predetermined level.

The level of absorptive capacity can be determined based on acombination of indices including, for example, K_(m), V_(max), andI_(sc).

The predetermined reference value can be established by a person skilledin the art. For instance, the predetermined reference value can beestablished by measuring the levels of the absorptive capacity of saidnutrient or electrolyte in normal small intestine epithelial tissuesthat do not have injury to the mucosa (such as villous cells, cryptcells, enterocytes, and intestinal progenitor cells). For anotherinstance, the predetermined reference value can be established bymeasuring the levels of the intestinal absorptive capacity of saidnutrient or electrolyte in a normal population who do not have injury tosmall intestine epithelial cells (such as villous cells, crypt cells,enterocytes, and intestinal projenitor cells).

In another embodiment, the subject screening method comprises:

a) determining function of small intestine tissue having injury in themucosa;

b) contacting candidate nutrient or electrolyte with the small intestinetissue;

c) determining the function of the small intestine tissue after thesmall intestine tissue is contacted with the candidate nutrient orelectrolyte; and

d) selecting the candidate nutrient or electrolyte if said candidatenutrient or electrolyte improves small intestine function.

In another embodiment, the subject screening method comprises:

a) determining small intestine function of a subject with injury tosmall intestine mucosa;

b) administering, via an enteral route, a candidate nutrient orelectrolyte to the subject;

c) determining the small intestine function of the subject after thecandidate nutrient is administered; and

d) selecting the candidate nutrient or electrolyte if said candidatenutrient or electrolyte improves small intestine function.

In certain embodiments, small intestine function is improved if theadministration of the candidate nutrient or electrolyte decreasesparacellular permeability, enhances small intestine barrier function.Also, small intestine function is improved if the enteral administrationof the candidate nutrient or electrolyte prevents or treats diarrhea,and/or prolongs survival.

In certain embodiments, the nutrient and electrolyte that improves smallintestine function of a subject with injury to small intestine mucosacan be selected using the methods as illustrated in the Examples,specifically, Examples 15-17.

Suitable candidate electrolytes include, for example, Na⁺, K⁺, HCO₃ ⁻,Cl⁻, Mg²⁺, Ca²⁺, Fe²⁺ and/or Zn²⁺.

Suitable candidate nutrients include essential and non-essential aminoacids selected from, for example, isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan, valine, histidine,tyrosine, selenocysteine, alanine, arginine, aspartate, cystein,glycine, proline, serine, asparagine, and pyrrolysine. Suitablecandidate nutrients may also include fatty acids, saccharides (e.g.,monosaccharides, di-saccharides, and oligosaccharides), eletrolytes, andvitamins.

Candidate nutrients may also include non-natural amino acids, such asfor example, ornithine, citrulline, hydroxyproline, homoserine,phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, γ-aminobutyric acid, ε-amino hexanoic acid, 6-amino hexanoic acid, 2-aminoisobutyric acid, 3-amino propionic acid, norleucine, norvaline,sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τ-butylalanine,phenylglycine, cyclohexylalanine, and β-alanine.

In a further embodiment, the selection of nutrients and electrolytesalso depends on, at least in part, the IR dosages received by thesubject, radiation sources, the body part being irradiated, and/or thetime that has elapsed after radiation; the type of chemotherapeuticagents, the dosage, and/or the time that has elapsed after chemotherapy;and the dosages of proton therapy received by the subject, and/or thetime that has elapsed after proton therapy.

The subject screening assays can be performed utilizing a combination oftechniques well known in the art, including but not limited to, Ussingchamber studies, cytology, immunohistochemistry, Western blots,enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction(PCR), ion flux experiments, immunoprecipitation, immunofluorescence,radioimmunoassay, and immunocytochemistry.

Specifically, the ingredients can be chosen based on their ability to beabsorbed by the small bowel mucosa of the patient, as determined byin-situ or isolated bowel preparations, using technologies such asUssing Chambers to measure the absorptive capacity of the smallintestine for such ingredient.

Formulations and Administration

The subject invention provides for therapeutic or pharmaceuticalcompositions comprising a therapeutically effective amount of thesubject composition and, optionally, a pharmaceutically acceptablecarrier. Such pharmaceutical carriers can be sterile liquids, such aswater. The therapeutic composition can also comprise excipients,adjuvants, flavoring agents, etc that do not affect the health orfunction of the injured small intestine epithelium, particularly in thevillous region and brush border. In an embodiment, the therapeuticcomposition and all ingredients contained therein are sterile.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin. Such compositions contain a therapeuticallyeffective amount of the therapeutic composition, together with asuitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the enteralmode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients, e.g.,compound, carrier, or the pharmaceutical compositions of the invention.

In one embodiment, the pharmaceutical pack or kit further comprisesinstructions for administration, for example, with respect to effectivetherapeutic doses, and/or the timing of administration with referenceto, for example, the elapse time from the exposure to radiation,chemotherapy, or proton therapy. In one embodiment, the therapeutic doseof the composition is determined based on the extent of injury to thesmall intestine mucosa. For instance, with regard to subjects thatreceive, or are about to receive radiation, the therapeutic dose of thecomposition is determined based on radiation sources, the body partbeing irradiated, and/or the time that has elapsed after radiation. Withregard to subjects that receive, or are about to receive chemotherapy,the therapeutic dose of the composition is determined based on the typeof chemotherapeutic agents, the dosage of chemotherapeutic agent, and/orthe time that has elapsed after chemotherapy. With regard to subjectsthat receive, or are about to receive proton therapy, the therapeuticdose of the composition is determined based on the dosages of protontherapy received by the subject, and/or the time that has elapsed afterproton therapy.

Materials and Methods

Experimental Animals

To study active HCO₃ ⁻ secretion, 8-week-old, non-irradiated andirradiated, male BALB/c mice were obtained from the National CancerInstitute. Mice were randomly divided into groups, and abdomens wereirradiated according to the gastrointestinal acute radiation syndrome(GI ARS) model with a Shepherd Mark-I, using a ¹³⁷Cs source deliveringγ-irradiation at 1.84 Gy/min. Radiation was given as a single fraction.The GI ARS model will achieve maximum radiation damage to intestinaltissues, and mimics intestinal injury during radiation therapy of pelvicor abdominal tumors.

Changes in short circuit current (I_(sc)), both as a function of timefollowing radiation and with increasing doses of radiation, wereexamined to determine the earliest time and the minimum radiation doserequired to produce significant changes in I_(sc). These studies wereapproved by the University of Rochester Animal Care and Use Committee.

Ion Flux Studies

Following exsanguinations, jejunal segment was obtained by excluding thedistal 12 cm of small intestine adjacent to the caecum. This segment waswashed and flushed in ice-cold Ringer's solution before the mucosa wasstripped from the underlying muscular layers (Zhang, Ameen et al. 2007).The mucosa was mounted between the 2 halves of an Ussing-type Lucitechamber with an area of 0.30 cm² (P2304, Physiologic instruments, SanDiego, Calif. 92128 USA), and electrical parameters were recorded usinga voltage/current clamp device (VCC MC-8, Physiologic instruments, SanDiego, Calif. 92128 USA) (Vidyasagar et al. 2005; Vidyasagar et al.2004; Zhang et al. 2007; Vidyasagar and Ramakrishna 2002).

TABLE 1 Compositions of solutions Ionic HCO₃ ⁻ HCO₃ ⁻- & compo- RegularHCO₃ ⁻ Na⁺ free Cl⁻-free free Cl⁻- free sition ringer free solutionsolution (UB) (UB) Na⁺ 140 140 — 140 140 140 Cl⁻ 119.8 119.8 119.8 —119.8 — K⁺ 5.2 5.2 5.2 5.2 5.2 5.2 HCO₃ ⁻ 25 — 25 25 — — HPO₄ ⁻ 2.4 2.42.4 2.4 — — H₂PO₄ ⁻ 0.4 0.4 0.4 0.4 — — Ca²⁺ 1.2 1.2 1.2 1.2 1.2 1.2Mg²⁺ 1.2 1.2 1.2 1.2 1.2 1.2 SO₄ ²⁻ — — — 1.2 2.4 2.4 Gluconate — — — —— — Cyclamide — — — 1.2 0.4 5.2 Isethionate — 25 — 115 25 140 NMDG — —140 — — — HEPES — — — — 0.1 0.1 Note: Values are in mM. Ionic solutionswere used for ion-substitution experiments. pH of all solutions were at7.4. H₂SO₄ was used to adjust the pH to 7.4 in Cl⁻-free solution, and inall others, HCl was used. Abbreviations: UB, unbuffered solutionIntestinal preparations were bathed bilaterally in a regular Ringer’ssolution (Table 1), containing 8 mM of glutamine and gassed with amixture of 95% oxygen (O₂) and 5% carbon dioxide (CO₂).Measurement of HCO₃ ⁻ Movement

A Bi-burette TIM 856 (Radiometer Analytical SAS, Villeurbanne, France)was used to measure HCO₃ ⁻ secretion in stripped jejunal sheets(Vidyasagar et al. 2005; Vidyasagar et al. 2004; Zhang et al. 2007).Automated pumps maintained a constant pH for luminal solution throughthe addition of 0.01 μl of 0.025 M sulfuric acid (H₂SO₄).Standard-to-stat pH calibration was established by adding a knownquantity of H₂SO₄ to a weak buffering solution, which contains anincreasing concentration of HCO₃ ⁻ to produce a linear titration curve.

Jejunal tissues were exposed to a buffered solution on the bath side(serosal side), while the luminal side was exposed to an HCO₃ ⁻ free,low-buffered solution (0.1-mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, pH 7.4).The HCO₃ ⁻ secretion was equivalent to the amount of acid added to theluminal solution to maintain the pH at 7.4 (or the stat pH). Allexperiments were performed under voltage-clamp conditions. HCO₃ ⁻-freesolution was gassed with 100% O₂ and HCO₃ ⁻-containing solution wasgassed with 95% O₂ and 5% CO₂. The HCO₃ ⁻ secretion was expressed as μeqh⁻¹·cm⁻² (Vidyasagar et al. 2005; Vidyasagar et al. 2004; Zhang et al.2007).

After the tissue was mounted, HCO₃ ⁻ secretions were initially presentin the absence of bath HCO₃ ⁻, but rapidly fell towards 0 within 20-30minutes. If bath HCO₃ ⁻ was not present during the titration, the HCO₃ ⁻secretion remained close to 0. Presence of HCO₃ ⁻ in the bath solutionresulted in a rapid increase in HCO₃ ⁻ secretions, which remainedconstant for at least 2 hours (Vidyasagar et al. 2005; Vidyasagar et al.2004; Zhang et al. 2007). When inhibitors were added to the mucosalsolution, the pH was adjusted and allowed to equilibrate for 30 minutes,until a steady rate of HCO₃ ⁻ secretion was observed. When the inhibitorwas added to the bath side, the tissue was also equilibrated for 30minutes to achieve a steady rate of HCO₃ ⁻ secretion (see Table 3).

All experiments were performed during the initial 1-hour steady-stateperiod. 1 tissue from each animal was used for each experiment; only 1experimental condition was studied with each tissue sample. Allexperiments were repeated for at least 4 times.

Immunohistochemistry

Frozen tissue slices from both non-irradiated and irradiated mice wereimmunofluorescence-stained using an anti-NBCe1-A/B antibody (Bevensee,Schmitt et al. 2000). NBCe1-A/B is a polyclonal antibody raised againstthe carboxy terminus, common to both sodium bicarbonate cotransporters(NBCe1-A and NBCe1-B). The immunostaining procedure was done on day 6post-irradiation. Isolated tissues were washed in ice-cold regularRinger's solution, embedded in frozen-section embedding medium, andplaced in liquid nitrogen; 6-μm sections were made in cryostat.

Western Blot Studies

Jejunal lysates were prepared from mucosal scrapings of non-irradiatedand irradiated mice. Tissues were analyzed for NKCC1 (Santa Cruz Calif.,USA), NBCe1-A/B (Mark Daniel Parker, Case Western Reserve UniversityMedical School, Cleveland, Ohio), and cystic fibrosis transmembraneconductance regulator (CFTR) (Santa Cruz Calif., USA) protein expressionby Western blots (Bevensee et al. 2000).

Mucosal scrapings were lysed in a triacylglycerol hydrolase buffercontaining 25-mM HEPES; 10% glycerol; and 1% Triton X-100 (polyethyleneglycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether) containing a proteaseinhibitor mixture with (10-mM iodoacetamide, 1-mM phenylmethylsulphonylfluoride, and 2-μg·ml⁻¹ leupeptin) at pH 7.4 (All chemicals wereobtained from Sigma-Aldrich Co., USA unless otherwise stated). Theprotein concentration was determined using the Bradford assay.Equivalent loads of proteins from irradiated and non-irradiated sampleswere analyzed using sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). NKCC1, NBCe1-A/B, and CFTR proteins weredetected using affinity-purified polyclonal antibodies.

Statistics

Results are presented as mean±standard error of mean. Statisticalanalysis was performed in 2 steps: 1) overall difference was testedusing analysis of variance (ANOVA) (or its non-parametric equivalentKruskal-Wallis); and 2) Bonferroni-adjusted P-values were computed forall pair-wise comparisons.

EXAMPLES

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Irradiation Increases Net Anion Secretion

This Example shows that irradiation increases net anion secretion, andcauses greater loss of villous epithelial cells as compared to cryptcells. Specifically, small intestine epithelial tissues were obtainedfrom mice that received 12 Gy irradiation and anion secretion wasexamined using Ussing chamber studies. Transepithelial I_(sc), anindicator of anion secretion, was measured on day 1, 2, 3, and 4.

As shown in FIG. 1A, maximal increase in transepithelial I_(sc) wasobserved at 48 hr post irradiation, as compared to non-IR exposedtissues and IR-exposed tissues 24 and 72 hrs post irradiation (FIG. 1A).This significant increase in I_(sc) at the end of 48 hrs indicates thatirradiation disrupts the fine balance between absorption and secretion.In comparison, I_(sc) recorded at the end of 48 hrs and 72 hrs is lowerthan that of non-IR mice tissues.

Histopathology sections also showed a greater loss of villous epithelialcells as compared to crypt cells due to irradiation. Whilehistopathology sections taken before 48 hrs showed minimal villousdamage and little or no crypt cell damage, histopathology sections takenon day 3 and 4 showed extensive damage in crypt and villous cells.Particularly, villous cells became almost completely depleted after day3. The loss of crypt cells was also observed, as evidenced by a failureto stimulate anion secretion in response to a secretory stimulus at 72and 96 hr post IR (FIG. 1A). At high doses of IR, there are insufficientcrypt stem cells, which mature and differentiate to form villousepithelial cells.

FIG. 1B shows that irradiation increased transepithelial conductance(FIG. 1B). Transepithelial conductance (S), a composite of transcellularand paracellular conductance, was measured by Ussing chamberexperiments.

Based on Ohms law 1/S=R, the increase in transepithelial conductanceindicates a reduction in transepithelial resistance (TER or R). Micesmall intestine has low-epithelial resistance. The electrical resistanceof the paracellular route is much lower than that of the transcellularresistance⁶⁵⁻⁶⁷ The paracellular route and the transcellular route arein parallel as shown by1/TER=(1/R_(transcellular))+(1/R_(paracellular)); hence, the measuredTER largely reflects paracellular resistance.

Example 2—Irradiation Causes a Dose-Dependent Increase in Short CircuitCurrent (I_(SC))

This Example reveals that irradiation causes a dose-dependent increasein short circuit current, indicating increased electrogenic anionsecretion. Briefly, mice that received 0, 1, 3, 5, 7, 9 or 12 Gyirradiation were sacrificed on day 4. FIG. 2 showed significant increasein I_(sc) in mice tissues irradiated at 3, 5 & 7 Gy, as compared to thatof those irradiated at 0 and 1 Gy (.*.p<0.001). Compared to miceirradiated at 3, 5 and 7 Gy, decreased I_(sc) was observed in micetissues irradiated at 9 & 12 Gy (*.*.p<0.01, FIG. 2 ). Irradiation atbetween 1 and 3 Gy resulted in the highest increase in I_(sc) andminimal histopathological changes.

In addition, irradiation causes changes in I_(sc) over time. Of micesacrificed on 0, 1, 2, 3, 4, 5, 6 or 7 days, the highest increase inI_(sc) was observed on day 5 and 6 post-IR (FIG. 3A). To determine themaximal increase in I_(sc) as a function of time, mice were irradiatedat 3 Gy and sacrificed on 0, 1, 2, 3, 4, 5, 6, 7, 9, 11, and 14 days torecord electrical parameters. Kruskal-Wallis (P<0.001). Post-hocanalysis showed the maximal increase in I_(sc) on day 6post-irradiation.

As shown in FIG. 3A, I_(sc) recorded on post-irradiation days 1 and 2showed little statistical differences. However, I_(sc) recorded ontime >2 days post-irradiation showed significant differences whencompared today 0 (.*. P<0.01). Among I_(sc) recorded on days 4, 5, 6,and 7, little significant difference was observed. I_(sc) recorded ondays 9 and 10 post-irradiation was also not significantly different fromthat recorded on day 7 post-irradiation (*.*. P═_(NS)). Although I_(sc),showed a significant decrease beyond day 6, it continued to stay at anelevated level on day 14 and even 2 years post-irradiation in mice whoreceived IR at 3-Gy (4.8±0.5 μeq·h⁻¹·cm⁻²). FIG. 3A shows that maximalincrease in I_(sc) occurred on day 6 in mice irradiated at 3 Gy.

The observed increase in I_(sc) post irradiation is largely due to a netincrease in electrogenic anion secretion. There are three possiblemechanisms for I_(sc) increase: 1) increased electrogenic anionsecretion (e.g., Cl⁻ and/or HCO₃ ⁻); 2) increased electrogenic Na⁺absorption; or 3) increased electrogenic K⁺ absorption. It is unlikelythat irradiation causes increased electrogenic Na⁺ absorptive process inmouse small intestine. In addition, as irradiation causes diarrhea,which results in K⁺ loss and not K⁺ absorption, the increase in I_(sc)cannot be due to increased K⁺ absorption.

Example 3—Decrease in Na⁺ and Cl⁻ Absorption

This Example shows that irradiation decreases Na⁺ and Cl⁻ absorption. Asshown in Table 2, Ussing chamber flux studies using ²²Na-substitutionrevealed that there is a net absorption of Na⁺ in non-IR (0 Gy) mice(Table 2), as the mucosal to serosal flux (J_(ms)) outperforms serosa tomucosa flux (J_(sm)). Irradiation decreases J_(ms) in a dose-dependentmanner, and results in decreased net Na⁺ absorption (J_(Net)Na). J_(sm)far exceeds J_(ms) at doses 7 and 9 Gy, causing net secretion. Inaddition, mice stool samples became loose or poorly formed at high doseirradiation, further evidencing decreased absorption and increasedsecretion of electrolytes. Similarly, net Cl⁻ absorption also decreasedas IR doses increased. Net Cl⁻ secretion was observed at 9 Gy. Decreasein Cl⁻ absorption was due to decrease in J_(ms)Cl⁻.

TABLE 2 Unidirectional and net flux of Na⁺ and Cl⁻ (J_(Net) = J_(ms) −J_(sm)) Na Flux IR Cl Flux IR Gy Jms Jsm Jnet Gy Jms Jsm Jnet ACTIVE 016.4 ± 0.9  7.1 ± 0.8 9.8 ± 0.8 0 17.3 ± 1.1  7.1 ± 0.6 10.2 ± 0.8 4365479 1 15.6 ± 1    7.1 ± 1.1 8.6 + 0.9 1 12.8 ± 0.9  7.6 ± 1.3 5.2 ±0.9 3 6.8 ± 0.7 3.5 ± 0.3 2.6 ± 0.5 3 16.8 ± 0.7  9.3 ± 0.8 7.5 ± 0.8 55.2 ± 0.6 4.8 ± 0.4 0.4 ± 0.2 5 14.2 ± 1.2  10.3 ± 0.9  3.9 ± 0.7 7 4.8± 0.4 5.4 ± 0.4 −0.6 ± 0.3   7 7.1 ± 0.5 6.9 ± 0.3 0.3 ± 0.2 9 4.3 ± 0.74.7 ± 0.6 −0.4 ± 0.2   9 −9.2 ± 0.8   0.1 ± 0.1 −9.3 ± 0.9  

Example 4—Irradiation Causes Increased Paracellular Permeability

This Example shows that irradiation results in the loss of smallintestine lining mucosa, leading to impaired small intestine barrierfunction. This increased small intestine permeability gives intestinalcomensal bacteria, peptides and toxins easier access to systemiccompartments, thereby causing endotoxemia. As shown in FIG. 4A,irradiation increases plasma endotoxin levels as measured by thetachypleus amebocyte lysate kit.

Irradiation also increased Cl⁻ & Na⁺ (PCl/PNa) permeability, asindicated by the changes of dilution potential determined in Ussingchamber studies. The use of dilution potential as the indicator ofmembrane permeability is based on the below principles. Specifically, anintact semi-permeable membrane, such as small intestine mucosa,maintains the electrochemical potential gradient artificially generatedby bathing mucosal and serosal side solutions with different ionicstrength. A leaky membrane that allows easy diffusion across themembrane, however, has diminished membrane electrochemical potential.Thus, the higher the permeability across the membrane, the lower thepotential gradient is. A freely permeable membrane has a relativepermeability of Cl⁻ and Na⁺ (PCl/PNa) at 1, which indicates a completeloss of selectivity.

In non-IR mice, the membrane selectivity is preserved and Na⁺ is morepermeable than Cl⁻ across the membrane. Irradiation decreased membranedilution potential. Particularly, Na⁺ and Cl⁻ became equally permeableacross the membrane at 7 Gy, indicating a significant loss ofselectivity (FIG. 4B). The increase in electrolyte permeability due toirradiation is consistent with the increase in plasma endotoxin levelsshown in FIG. 4A. Monitoring changes in membrane permeability can beused as a sensitive tool to monitor improvement in mucosal barrierfunction by the subject oral radiation diet.

Example 5—Increase in Levels of Inflammatory Mediators Due toIrradiation

Levels of inflammatory mediators in IR-exposed and non-IR exposed micewere measured using LUMINEX multiplex bead array techniques. As shown inFIG. 5 , irradiation increased the production of IL1-β, TNF-α and MIP-α(FIG. 5 ).

Example 6—Decreased in Anion Secretion Due to Irradiation isNKCC1-Dependent and CFTR-Dependent

This Example shows that anion secretion under irradiation isNKCC1-dependent and CFTR-dependent. To determine the contribution ofNKCC1 to basal I_(sc), 100 μM bumetanide (Sigma-Aldrich Co., USA) wasadded to the bath solution. FIG. 3C showed a bumetanide-inhibitablecurrent in irradiated tissues (5.5±0.5 μeq·h⁻¹·cm⁻² vs. 0.6±0.1μeq·h⁻¹·cm⁻²), but not in 0-Gy mice (1.6±0.2 μeq·h⁻¹·cm⁻² vs. 0.9±0.1μeq·h⁻¹·cm⁻²). In addition, cAMP-stimulation caused an increase inI_(sc) in both 0-Gy (1.6±0.2 μeq·h⁻¹·cm⁻² vs. 6.9±0.6 μeq·h⁻¹·cm⁻²,P<0.001) and 3-Gy irradiated mice (5.5±0.5 μeq·h⁻¹·cm⁻² vs. 7.3±0.5μeq·h⁻¹·cm², P<0.05).

In addition, forskolin (Sigma-Aldrich Co., USA)-stimulated I_(sc) wasabated by bumetanide in 3 Gy (7.3±0.5 μeq·h⁻¹·cm⁻² vs. 0.4±0.1μeq·h⁻¹·cm⁻²), but not in 0 Gy (6.9±0.6 μeq·h⁻¹·cm⁻² vs. 1.3±0.2μeq·h⁻¹·cm⁻²). This indicates greater NKCC1-independent anion secretionwithout irradiation (P<0.05).

The results also showed that anion secretion under irradiation isCFTR-dependent. To determine whether the bumetanide-insensitive portionof I_(sc) occurs via an apical membrane anion channel, a non-specificanion channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid(Sigma-Aldrich Co., USA) (10 μM NPPB), and a specific cystic fibrosistransmembrane conductance regulator (CFTR) blocker (100 μMglibenclamide, Sigma-Aldrich Co., USA) were applied.Bumetanide-insensitive I_(sc) in 0-Gy mice was abolished by mucosaladdition of a non-specific anion channel blocker (NPPB) (0.1±0.01μeq·h⁻¹·cm⁻²) and glibenclamide (0.1±0.01 μeq·h⁻¹·cm⁻²). This indicatesthat anion secretion occurs via an anion channel or CFTR (FIG. 3B).

Example 7—Decrease in HCO₃ ⁻ Secretion Due to Irradiation

Infective diarrhea, such as cholera, results in the loss of HCO₃ ⁻-richfluid in stool and leads to metabolic acidosis. This Example shows that,in contrast to infective diarrhea, IR induced increased Cl⁻ secretionand decreased HCO₃ ⁻ secretion.

To determine the contribution of Cl⁻ to net anion secretion, a blockerfor Cl⁻ uptake into the cell (Na—K-2Cl cotransport blocker) wasemployed. Addition of 10 μM bumetanide abolished almost all of theI_(sc) associated with IR, suggesting that IR-induced anion secretion isprimarily due to increased Cl⁻ secretion and such increase isNKCC1-dependent (FIGS. 3A-C).

pH stat experiments confirmed that IR reduced HCO₃ ⁻ secretion (Table3). HCO₃ ⁻ secretion was abolished when Na⁺ in the serosal bathingsolution (bath) was replaced with an impermeable cation NMDG, indicatingthat transport of HCO₃ ⁻ into the cell at basolateral membrane is bathNa⁺ dependent. Similar experiments were repeated in 5 Gy, day 6 post IRmice. In the presence of bath Na⁺, HCO₃ ⁻ secretion was significantlylower.

Immunofluorescence staining of frozen tissue slices obtained from bothnon-IR and IR mice was performed using NBCe1a/b antibodies (FIGS. 6B-E).NBCe1a/b antibody-specific staining showed that NBCe1a/b was expressedin the villous epithelial cells, but not in the crypt cells.Immunostaining of tissues from IR mice showed that NBCe1a/b antibodieswere not recognized either in the villous or in the crypt. Tissues frommice irradiated with 3 Gy (IR) failed to express NBCe1-A/B-specificstaining pattern either in the villous or in the crypt. Decreased HCO₃ ⁻secretory function at high doses of IR is due to the loss of villousepithelial cells. Monitoring changes in Na⁺ and HCO₃ ⁻ secretion can bea sensitive tool to monitor improvement of mucosal barrier function bythe subject oral radiation diet.

HCO₃ ⁻ Secretion Under Irradiation is NKCC-Independent

To determine if HCO₃ ⁻ contributed to anion secretion, experiments wereperformed in the absence of bath Cl⁻. An increase in I_(sc) secondary toirradiation or forskolin-stimulation was considered to be contributed byHCO₃ ⁻. The results showed that HCO₃ ⁻ secretion under irradiation isnot bath Cl⁻-dependent; therefore, under irradiation, HCO₃ ⁻ secretiondoes not involve the Cl⁻—HCO₃ exchange transporter (AE1) in the apicalmembrane. In Cl⁻-free solution, basal (1.0±0.2 μeq·h⁻¹·cm⁻² vs 0.3±0.1μeq·h⁻¹·cm⁻²; P=ns) and forskolin-stimulated (1.7±0.2 μeq·h⁻¹·cm⁻² vs.0.3±0.1 μeq·h⁻¹·cm⁻²; P<0.001) I_(sc) was lower in 3-Gy irradiated mice(FIG. 3D). Forskolin-stimulated L was higher in 0 Gy than in 3 Gy(P<0.001), indicating a decrease in HCO₃ ⁻ secretion due to irradiation.

To ascertain if NKCC1 mediated HCO₃ ⁻ movement under basal andforskolin-stimulated conditions, bumetanide was added to the bath sideof tissues equilibrated in Cl⁻-free solution on both sides. The resultsshowed that bumetanide did not inhibit basal and forskolin-stimulatedincrease in I_(sc); this lack of inhibition indicates aNKCC1-independent mechanism for HCO₃ ⁻ uptake at the basolateralmembrane.

HCO₃ ⁻ Secretion Under Irradiation is Lumen Cl⁻-Independent

Direct measurement of HCO₃ ⁻ secretion in irradiated mice showed reducedHCO₃ ⁻ secretion compared to non-irradiated mice (0.8±0.2 μeq·h⁻¹·cm⁻²vs. 6.7±0.2 μeq·h⁻¹·cm⁻²). HCO₃ ⁻ secretion in irradiated mice wasunaltered by removal of lumen Cl⁻ (Table 3). The mucosal addition ofNPPB (0.2±0.01 μeq·h⁻¹·cm⁻²) and glibenclamide (0.11±0.1 μeq·h⁻¹·cm⁻²),but not DIDS, ended HCO₃ ⁻ secretion in irradiated mice. This indicatesthat HCO₃ ⁻ secretion is mediated by an anion channel (CFTR channel),not via Cl⁻—HCO₃ ⁻ exchange (FIG. 19B).

In comparison, HCO₃ ⁻ secretion in non-irradiated mice is both lumenCl⁻-dependent and Cl⁻-independent. Transepithelial electricalmeasurements indicated electrogenic HCO₃ ⁻ secretion; however, this doesnot indicate whether HCO₃ ⁻ secretion was channel-mediated and/or viaelectroneutral Cl⁻—HCO₃ ⁻ exchange.

pH-stat experiments were performed in the absence of lumen Cl⁻ to studyCl⁻—HCO₃ ⁻ exchange in non-irradiated mice. In a lumen Cl⁻-freesolution, HCO₃ ⁻ secretions were lower (4.5±0.1 μeq·h⁻¹·cm⁻², P<0.01).This indicates that basal HCO₃ ⁻ secretion in non-irradiated mice ispartly lumen Cl⁻-dependent and partly Cl⁻-independent (Table 3). Theaddition of 100 μM 4,4-diisothiocyano-2,2′-stilbene disulfonic acid(DIDS) (Sigma-Aldrich Co., USA) partially inhibited HCO₃ ⁻ secretion(P<0.001), and such inhibition was similar to that observed with lumenCl⁻ removal.

Forskolin Stimulated Lumen Cl⁻-Independent HCO₃ ⁻ Secretion

For 0-Gy mice, addition of forskolin to the bath solution showedsignificant increases in basal HCO₃ ⁻ secretion (P<0.001) that was notaltered by lumen Cl⁻ removal (8.4±0.4 μeq·h⁻¹·cm⁻² vs. 8.7±0.4μeq·h⁻¹·cm⁻²; n=6). NPPB abolished forskolin-stimulated HCO₃ ⁻ secretion(0.2±0.01 μeq·h⁻¹·cm⁻²; n=6); this indicated a role for an anion channelin cAMP-stimulated HCO₃ ⁻ secretion.

cAMP-Stimulated HCO₃ ⁻ Secretion is NKCC1-Independent

To determine if cAMP-stimulated HCO₃ ⁻ secretion required an apical CFTRchannel, glibenclamide was added to the luminal side. Glibenclamideinhibited (0.1±0.1 μeq·h⁻¹·cm⁻²) HCO₃ ⁻ secretion (Table 3 and FIG.19A), indicating that cAMP not only inhibits the basal Cl⁻—HCO₃ ⁻exchange component of the net HCO₃ ⁻ secretion, but also induces anapical anion channel-mediated HCO₃ ⁻ secretion. Forskolin stimulation inirradiated mice showed little increase compared to basal HCO₃ ⁻secretion (0.6±0.2 μeq·h¹·cm⁻² vs. 0.78±0.2 μeq·h⁻¹·cm⁻²). This alsoindicates a lumen Cl⁻-independent HCO₃ ⁻ secretion or the inhibition ofCl⁻—HCO₃ ⁻ exchange.

Transepithelial electrical measurements showed that decrease in HCO₃ ⁻movement was also NKCC1-independent. HCO₃ ⁻ secretion, which was minimalin irradiated mice under both basal and forskolin-stimulated conditions,was unaffected by the addition of bumetanide (Table 3). Similarly, innon-irradiated mice, bumetanide did not alter forskolin-stimulated HCO₃⁻ secretion, indicating that the cAMP-stimulated HCO₃ ⁻ secretion isNKCC1-independent (8.4±0.4 μeq·h⁻¹·cm⁻² vs. 8.6±0.4 μeq·h⁻¹·cm⁻²) (Table3 and FIG. 3B).

HCO₃ ⁻ Secretion is Bath Cl⁻-Independent

Transport processes requiring bath Cl⁻ for basolateral HCO₃ ⁻ uptake areshown in FIG. 3B. The results showed that bumetanide did not alter thecAMP-stimulated HCO₃ ⁻ secretion. This indicated that Cl⁻—HCO₃ ⁻exchange (AE2) transporter is inhibited under irradiation. Removal ofbath Cl⁻ can also inhibit NKCC1 and AE2-linked HCO₃ ⁻ uptake (Table 3).Removal of Cl⁻ from the bath solution did not alter HCO₃ ⁻ secretion(6.7±0.3 μeq·h⁻¹·cm⁻² vs. 7.1±0.6 μeq·h⁻¹·cm⁻²).

HCO₃ ⁻ Secretion is Bath Na⁺-Dependent

Transport processes for the Na⁺-coupled, basolateral, HCO₃ ⁻ entry areshown in FIG. 3B. FIGS. 3C and 3D indicated that NKCC1 does not affectHCO₃ ⁻ secretion. Addition of 1 mM3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE694)to the bath side eliminated HCO₃ ⁻ uptake via NHE1 coupled to Cl⁻—HCO₃ ⁻exchange. Counillon, Scholz et al. (1993) also described that 1 mM3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE694)could inhibit Na⁺—H⁺ exchange (NHE1).

HOE694 did not inhibit cAMP-stimulated HCO₃ ⁻ secretion (8.4±0.4μeq·h⁻¹·cm⁻² vs. 7.2±0.9 μeq·h⁻¹·cm⁻²). Replacing bath Na⁺ withN-methyl-D-glucamine (NMDG) abolished forskolin-stimulated HCO₃ ⁻secretion (8.4±0.4 μeq·h⁻¹cm⁻² vs. 0.3±0.01 μeq·h⁻¹ cm⁻²) innon-irradiated mice, which indicates a Na⁺— coupled HCO₃ ⁻ cotransport(NBC) (FIG. 3E).

TABLE 3 HCO₃ ⁻ secretion measured in the jejunum of non-irradiated (0Gy) and irradiated (3 Gy) mice. Lumen Cl⁻ 100 μM 100 μM solutioncontaining Cl⁻-free DIDS glibenclamide 0 Gy 6.7 ± 0.3   4.5 ± 0.1^(‡)  4.4 ± 0.1^(‡)    0.5 ± 0.1   3 Gy 0.8 ± 0.2*  0.6 ± 0.1*^(ns) 0.9 ±0.2*^(ns)  0.1 ± 0.1^(ns) 0 Gy + forskolin 8.4 ± 0.4   8.7 ± 0.4    —0.1 ± 0.1   3 Gy + forskolin 0.6 ± 0.2   0.9 ± 0.2    — — 0 Gy + 8.6 ±0.4   8.4 ± 0.4    7.7 ± 0.4     0.5 ± 0.1   bumetanide 3 Gy + 0.8 ±0.14* 0.8 ± 0.1^(ns ) 0.7 ± 0.12^(ns ) 0.2 ± 0.1^(ns) bumetanide Note:Values represent mean ± SEM n = 6 tissues. *p < 0.001, comparisonbetween 0 Gy and 3 Gy group. ^(‡)p < 0.001 comparison between presencegroups. In bumetanide experiments in non-irradiated mice, the tissueswere treated with 10 mM forskolin. Abbreviations: ns, no significancebetween the groups; 4,4-diisothiocyano-2,2′-stilbene disulfonic acid,DIDS

For active HCO₃ ⁻ secretion at the apical membrane, there is a need forits basolateral uptake. Four known exchange mechanisms directly orindirectly involved with HCO₃ ⁻ movement at the basolateral membraneare: 1) Na⁺—K⁺-2Cl⁻ co-transport (Na⁺—K⁺-2HCO₃ ⁻) as a possibletransporter of HCO₃ ⁻; 2) Cl⁻ uptaken into the cell via NKCC1 isrecycled via basolateral Cl⁻—HCO₃ ⁻ exchange (AE2), resulting in netHCO₃ ⁻ uptake at the basolateral membrane; 3) Na⁺—H⁺ exchange extrudingprotons into intercellular space, resulting in decreased intracellularHCO₃ ⁻ concentration, which then stimulates apical electroneutralCl⁻—HCO₃ ⁻ exchange; and 4) Na⁺ coupled HCO₃ ⁻ cotransport. Thesetransporters may function as electroneutral or electrogenic, dependingon the number of HCO₃ ⁻ molecules transported per molecule of Na⁺ (FIG.3B).

In non-irradiated mice, HCO₃ ⁻ uptake occurs via a Na⁺-coupled HCO₃ ⁻cotransporter (NBCe1-A/B) located at the basolateral surface. Apicalexit occurs via an electroneutral Cl⁻/HCO₃ ⁻ exchange that is coupled toa Na⁺—H⁺ exchange and via CFTR (electrogenic anion secretion). Anincrease in intracellular cAMP, achieved by the addition of forskolin,results in increased Cl⁻ and HCO₃ ⁻ secretion with simultaneousinhibition of electroneutral Na⁺ and Cl⁻ absorption (Na⁺—H⁺ exchangecoupled to Cl⁻—HCO₃ ⁻ exchange). Cl⁻ uptake occurs via NKCC1, and HCO₃ ⁻uptake occurs via NBCe1-A/B; both Cl⁻ and HCO₃ ⁻ exit via CFTR at theapical surface.

In accordance with the subject invention, it has been discovered thatirradiation inhibits electroneutral Na⁺ and Cl⁻ absorption. Irradiationalso inhibits NBCe1-A/B, and such inhibition results in decreasing HCO₃⁻ uptake at the basolateral membrane and finally its exit at the apicalmembrane. Thus, irradiation results in electrogenic Cl⁻ secretion withselective inhibition of both electroneutral and electrogenic HCO₃ ⁻;secretion (FIG. 19B).

Irradiation caused increased NKCC-1 protein expression and decreasedNBCe1-A/B expression in the small intestine epithelium tissues.Irradiation also inhibits the apical Cl⁻—HCO₃ ⁻ exchange transporter(AE1). HCO₃ ⁻ secretion in radiation diarrhea is Na-dependent, but lumenCl⁻ independent and NKCC-1-independent. Cl⁻ transport under irradiationinvolves the basolateral NKCC-1 transporter, instead of a Cl⁻—HCO₃ ⁻exchange transporter (AE1).

As shown in FIG. 19B, irradiation also alters electrolyte (such as HCO₃⁻ and Cl⁻) transport in the gastrointestinal tract. Irradiated miceexhibited primarily Cl⁻ secretion, and minimal HCO₃ ⁻ secretion. It ispostulated that minimal HCO₃ ⁻ secretion due to irradiation is caused bythe inhibition of HCO₃ ⁻ absorption. In contrast, there is active Cl⁻ aswell as HCO₃ ⁻ secretion in secretagogue-induced diarrhea.

Example 8—Irradiation Causes Reduced Glucose Absorption

This Example shows that in IR-induced enteritis, there is adose-dependent decrease in glucose absorption. In addition, the presenceof unabsorbed glucose in the gut lumen can lead to osmotic diarrhea,further deteriorating the diarrheal conditions associated with IR. FIG.7A shows that irradiation causes a dose-dependent decrease in I_(sc).FIG. 7B shows that irradiation increases K_(m) in glucose transport in adose-dependent manner.

SGLT1 is a versatile transporter. SGLT1 maintains its function ininfective diarrhea, such as cholera. The preservation of SGLT1 functionin infective diarrhea has been used in oral rehydration therapy for Na⁺absorption.

To investigate SGLT-1 function and its effect on glucose absorptionfollowing irradiation, small intestine mucosa of Swiss mice was obtainedon day 6 after IR exposure at 0, 1, 3, 5, or 7 Gy. Glucose-stimulatedshort-circuit current (I_(sc)) was measured in an Ussing chamber tostudy the SGLT1 transport function. Survival studies were carried out in9-Gy TBI and 15.6-sub-TBI mice.

Specifically, 8-week-old Balb/c mice obtained from the NationalInstitutes of Health (NIH) were subject to 137Cs sub-total bodyirradiation (Sub-TBI) (one leg was protected from irradiation) andtotal-body irradiation (TBI).

In animal survival studies, mice were separated into 2 groups: 9-Gy TBIand 15.6-Gy Sub-TBI. Control mice were treated with normal saline;others were treated with 5% glucose. Gavage was used during theexperiment, and treatments were given on the first 5 days afterirradiation and every other day until 10 days after irradiation.

A Multichannel Voltage/Current Clamp (Physiological Instruments, SanDiego, Calif.) was employed in the Ussing chamber study. Mice jejunalsections, which were used for the mounting, were bathed inmodified-regular Ringer's solution, and gassed with 95% 02 & 5% CO2 tomeasure short I_(sc). All mice were sacrificed 6 days after irradiation.

To investigate SGLT-1 kinetics, the substrate (glucose) concentrationstarted at 0.05 mM and ended at 10 mM. Glucose was added at a ratestarting from 0.05 mM and progressed to 0.1 mM, 0.5 mM, and 1 mM. Theresults were analyzed with Origin 8 software (OriginLab Corp.,Northhampton, Mass.). I_(sc) was plotted into the Y-axis, and glucoseconcentration was plotted into the X-axis. The curve was fitted intoHill's equation.

To prepare Jejunal whole-cell lysates, mucosal scrapings of normal andirradiated mice were lysed in triacylglycerol hydrolase buffercontaining 25-mM HEPES, 10% glycerol, 1% Triton X-100, and a proteaseinhibitor mixture (10 mM iodoactamide, 1 mM phenylmethylsulphonylfluoride, and 2 μg ml-1 leupeptin, pH 7.4).

To prepare brush-border membrane vesicle lysates, mucosal scrapings ofnormal and irradiated mice were homogenized in a 2-mM Tris-HCl (pH7.1)/50-mM KCl/1M PMSF solution. The samples were spun down with acentrifuge at 8000 RPM and again at 13,000 RPM, respectively, and werethen homogenized again with a turberculin syringe (27G needle) andTEFLON homogenizer. The samples were then centrifuged at 4,000 RPM andagain at 15,000 RPM. The sample was resuspended with a proteaseinhibitor mixture (10 mM iodoactamide, 1 mM phenylmethylsulphonylfluoride, and 2 μg ml-1 leupeptin, pH 7.4) that contained regularRinger's solution.

Both protein concentrations of jejunal whole-cell lysates andbrush-border membrane vesicles were analyzed for SGLT-1 protein byWestern blots. Equivalent loads of protein from irradiated and controlsamples were analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). Proteins were transferred ontopolyvinylidene fluoride (PVDF) membranes, and SGLT-1 proteins weredetected using affinity-purified polyclonal antibodies.

The results, as shown in FIGS. 8-14 , demonstrate that: 1) irradiationreduces glucose-stimulated I_(sc) in a dose-dependent manner; 2) K_(m)values for glucose were (mM) 0.38±0.04, 0.49±0.06, 1.76±0.16, 1.91±0.3,2.32±0.4 in 0, 1, 3, 5, and 7 Gy, respectively; 3) V_(max) values forglucose were 387.4±16.2, 306.6±16.4, 273.2±14.9, 212.9±9.14, 188.1±9.12in 0, 1, 3, 5, and 7 Gy, respectively; 4) K_(m) and V_(max) valuesreturned to normal levels approximately 14 days after IR; 5) withholdingglucose intake for the first 10 days after irradiation increasedsurvival; 6) Western blot analysis of the SGLT-1 brush-border membraneshowed increased SGLT-1 protein levels as IR doses increased.

The increase in SGLT-1 K_(m) indicates a decrease in SGLT-1 affinity forglucose due to irradiation. The decrease in V_(max) indicates the lossof villous epithelial cells due to irradiation, as is also evidenced bythe histopathological examinations. The increase in protein levels inmice tissues treated with IR, as shown in Western blot analysis,indicates that SGLT1 transporters are expressed but non-functional.

The results also demonstrate oral glucose feeding results inmalabsorption of glucose and electrolytes, which leads to osmoticdiarrhea and, thus, increases IR-induced GI toxicity. In contrast,withholding glucose from oral feeding for first 14 days after IRprevents or mitigates symptoms of diarrhea and increases overallsurvival.

Example 9—Irradiation Causes Reduced Glutamine Transport

Although glutamine is a non-essential amino acid, it is the primarynutrient of the enterocytes, and is present in high concentrations inplasma (26%) and in skeletal muscle (75%). Glutamine levels decrease inpost-operative, trauma, or critical patients as the body's demand forglutamine increases. Thus, glutamine has been considered as important inthe normal functioning of the digestive, renal, immune and neuronalsystems.

This Example shows that irradiation causes a dose-dependent decrease inglutamine transport into the cells. At IR≥7 Gy, glutamine becomeslargely present in the gut lumen, thereby leading to osmotic diarrhea.Saturation kinetics of glutamine transporter showed an IR dose-dependentincrease in K_(m) (FIG. 15 ), suggesting decreased affinity of glutaminetransporters for glutamine.

Example 10—Irradiation Causes a Dose-Dependent Increase in LysineTransport

Addition of lysine to the small intestine lumen side causes an increasein I_(sc), suggesting electrogenic transport of lysine (FIG. 16 ).Tissues from non-IR mice showed a K_(m) of 1.16±0.04 mM, while 3 Gy IRtissues had a K_(m) of 0.27±0.01 mM. Unlike glucose and glutamine, theresults showed that irradiation increased lysine-transporter affinityfor lysine and, thus, increased lysine absorption.

Example 11—Effect of Oral Lysine Feeding on Survival of Mice

This Example shows that withholding non-absorbed nutrients from oralfeeding while selectively feeding absorbed nutrients prevents ormitigates diarrhea and increases survival after irradiation.

In the first series of experiments, glucose (10 mM i/m for 5 days andthen every alternate day) was orally administered to IR mice. Theresults show that glucose administration decreased overall survival(FIG. 17B). In comparison, lysine (20 mg/mice/day) was orallyadministered to IR mice for 5 days and thereafter every other day asgastric lavage. Mice treated with lysine showed increased survival whencompared to control groups (FIG. 17A). Thus, reducing or limiting oralintake of non-absorbed nutrients such as glucose with increased oralintake of absorbed nutrients such as lysine can prolong survival inirradiated patients.

Example 12—Changes in Ion Transport Protein Expression Levels Due toIrradiation

This Example illustrates changes in transport protein expression levelsdue to irradiation.

Specifically, tissues were harvested for Western blot on day 6post-irradiation. Western blot of ileal tissues, as shown in FIG. 18 ,revealed that irradiation from 1-5 Gy resulted in increased NKCC1protein levels; while such increase in NKCC1 expression decreased intissues received 7 Gy IR, as compared to tissues received 1-5 Gy IR (A).

NBCe1-A/B protein levels significantly decreased following irradiation,even at a dose as low as 1 Gy (B). CFTR protein levels in jejunumtissues significantly increased following 3 Gy irradiation, as comparedto 0 Gy jejunum tissues (C). NBCe1-A/B specific antibodies showedincreased expression levels in the jejunum compared to the duodenum,ileum, and colon in non-irradiated mice (D). Jejunum tissues had thehighest NBCe1-A/B protein levels, as compared to that in duodenum, ileumor colon (D). The changes in levels of transport protein correspond tothe observed functional changes following IR. The expression pattern ofthe transport proteins post-irradiation, as compared to that of non-IRtissues, can be used to monitor the effectiveness of the oral radiationdiet.

Example 13 Changes in Nutrient and Electrolyte Absorptive Capacity inMice with Injury to Small Intestine Mucosa

A similar pattern of alterations in small intestine absorptive capacityis observed in C57BL/6 mice treated with radiation, chemotherapy, andsuffering from inflammation in the small intestine. The radiation modelis constructed as described in Examples 1-12.

In a chemotherapy model, all mice are injected with a single dose of5-FU or cisplatin. In some mice, three days after the first injection, asecond dose of 5-FU or cisplatin is injected. Following each injection,transepithelial I_(SC), an indicator of net anion secretion, is measuredusing an Ussing chamber, at time points as indicated in FIG. 20 . Foreach measurement, a minimum of 32 tissues is examined.

The results show that there is a significant increase in net anionsecretion on day 3 in mice injected with a single dose of cisplatin(FIG. 20B) or 5-FU (FIG. 20A). Also, mice injected with a second dose ofthe chemotherapeutic agent exhibit a significantly higher increase innet anion secretion than that of mice receiving a single dose.

In a Crohn's disease model, mice are injected with anti-CD3 mAb (acuteinflammatory model to mimic conditions of Crohn's disease). There isalso a significant increase in net anion secretion (determined based onparacellular conductance) and paracellular permeability of the smallintestine. Also, alterations in nutrient and electrolyte absorptivecapacity are observed.

The alterations of absorptive capacity of nutrients are determined usingdisease models with injury to small intestine mucosa, i.e., theradiation model, chemotherapy model, and the Crohn's disease model.Specifically, a candidate nutrient is orally administered to controlmice and mice that received irradiation, chemotherapy, and anti-CD3 mAb,respectively. In addition, compositions containing various combinationsof the candidate nutrients are orally administered.

The candidate nutrients are selected from lysine, histidine, valine,leucine, phenylalanine, cysteine, tyrosine, arginine, isoleucine,threonine, glycine, alanine, methionine, tryptophan, proline, serine,asparagine, glutamine, aspartic acid, glutamic acid, and glucose.

To determine the absorptive capacity of each nutrient, bioelectricmeasurements are performed using an Ussing chamber. The measurementsinclude: a) the sodium coupled-amino acid current (I_(SC)) and changesin conductance, b) changes in saturation kinetics of each nutrient andchanges in the I_(SC) following the administration of each nutrient; andc) the electrolyte absorption studies using isotope flux studies in thepresence and absence of the specific candidate nutrient. The resultsshow that, in the radiation model, chemotherapy model, and the Crohn'sdisease model, there is a similar pattern of alterations in absorptivecapacity for all amino acids investigated and glucose. Specifically, theresults show that the oral administration of each of the following aminoacids selected from lysine, glycine, threonine, valine, tyrosine,aspartic acid, isoleucine, tryptophan, asparagine, and serine improvesmall intestine healing, reduces paracellular conductance (therebyimproving small intestine mucosal barrier mechanism), increasesabsorption of electrolytes, and/or improves survival in animals. Theresults also show that the oral administration of glucose and/orglutamine impairs small intestine mucosa barrier, and has adverseeffects on survival of mice in the radiation model, chemotherapy model,and the Crohn's disease model.

Example 14—Improvement of Small Intestine Function in Mice that haveReceived Chemotherapy

This Example shows that the therapeutic composition of the subjectinvention improves small intestine healing of mice that have receivedchemotherapy. Of all chemotherapy drugs studied, 5-FU shows maximumtoxicity to small intestine. Therefore, 5-FU is used to characterize thealterations of electrolyte and nutrient transport in the chemotherapymodel.

NIH Swiss mice were injected with 5-FU. Five or six days afterinjection, The intestinal tissues from the mice were isolated andstudied in an Ussing chamber, exposing to either Ringer solutions or thetherapeutic composition of the subject invention. The therapeuticcomposition contains lysine, glycine, threonine, valine, tyrosine,aspartic acid, isoleucine, tryptophan, asparagine, and serine; water;and therapeutically acceptable carriers, electrolytes, and bufferingagents. The therapeutic composition is slightly alkaline (pH 7.4). Thetherapeutic composition does not contain glucose, glutamine, ormethionine.

The results show that the therapeutic composition significantly improvessmall intestine function of mice that have received 5-FU. Specifically,the therapeutic composition significantly reduces the pathologicalincrease in transepithelial I_(SC) (FIG. 21A) and transepithelialconductance in the small intestine of the 5-FU injected mice.

Example 15—Determination of Changes in GI Function Due to Irradiation

The major GI function includes absorption of nutrients, electrolytes andwater, and such absorption occurs in well-differentiated and maturevillous epithelial cells. 80% of the fluid and electrolyte absorptionoccurs in the small intestine. As illustrated herein, IR results inselective loss of villous and/or crypt depending on the IR dose, andthereby leads to decreased absorption of Na⁺, Cl⁻ and nutrients. ThisExample illustrates experimental designs for determining alterations inGI function caused by various dosages over time IR.

Methods

C57BL/6 mice (8 weeks old, male) from NCI are used. Physicalobservations, cytology, immunohistochemistry, Western analysis, plasmasurrogate markers, and functional studies are determined as specificindices for IR-induced GI toxicity. Mice were randomly divided intogroups and the abdomen irradiated with a Shepherd Mark-I using a Cssource delivering IR at 1.84 Gy/min dose rate to the abdomen. Mice aresubject to IR at 0, 1, 3, 5, 7 and 9 Gy. Changes in glucose and aminoacid transport are examined on day 0, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 25 and 30 with 10 mice in each group. Plasma samples are collectedbefore harvesting the tissue. Ileum and jejunum tissues are harvestedfor histopathology, Western blot, immunohistochemistry and Ussingchamber studies (subjected to separate evaluation).

A) Determination of Functional Alterations in Electrolytes (Na⁺, Cl⁻ &HCO₃ ⁻)

This Example illustrates experimental designs for determining thealteration in transport protein function associated with electrolyteabsorption following R. The alterations in electrolyte transportfunction are then correlated to plasma markers, cytology, and physicalobservations such as daily activity, body weight, stool formation andfecal occult blood. Cytology examinations are performed using cryptassay, H&E staining, BrdU staining, immunohistochemistry and Westernblot analysis.

First, transepithelial flux of Na⁺ and Cl⁻ is examined in an Ussingchamber to evaluate electrolyte absorption after IR. Mice aresacrificed, and the changes in the basal ion transport in the non-IRmice and mice treated with IR at various doses are examined. Na⁺ and Cl⁻absorption is electroneutral in regular epithelium.

In this Example, isotope (²²Na and ³⁶Cl) substitution studies areperformed to determine the basal Na⁺ and Cl⁻ movement. Briefly, ²²Na and³⁶Cl are added either to the mucosal or to the serosal side. 0.5 mlsamples are collected from the cold side at the end of every 30 minutes.Unidirectional fluxes are calculated using standard formula, andexpressed as μmol·h⁻¹·cm⁻². Net flux (J_(Net)) is calculated as thedifference between J_(ms) and J_(sm) fluxes across tissue pairs.Experiments are performed under short-circuit conditions.

In addition, pH stat techniques are used to measure changes in HCO₃ ⁻secretion. As illustrated herein, IR decreased HCO₃ ⁻ secretion injejunum. HCO₃ ⁻ secretion is critical for acid base balance and acidneutralization in the upper segments of gut⁷²⁻⁷⁴. These experimentssuggest the possible mechanism of HCO₃ ⁻ secretion and indicate 1) lumenCl-dependent HCO₃ ⁻ secretion and 2) lumen Cl⁻-independent HCO₃ ⁻secretion in normal mice and in irradiated mice. Bicarbonate secretionis expressed as follows:

${{Total}{bicarbonate}{{secretion}( {\mu{eq}/h/{cm}^{2}} )}} = \frac{( {{D2} - {D1}} ) \times 0.025 \times 2 \times 60}{1.13 \times (t)}$where D2 and D1 are the difference between the total acid added betweentwo time points, 0.025 represents the normality of the acid added, 2 thevalency of H₂SO₄ and 60 represents the time in minutes to finallyexpress secretion per hour. 1.13 represents the surface area of thetissue used in the Ussing chamber and t time. HCO₃ ⁻ secretion studiedusing pH stat technique will complement transepithelial Na⁺ and Cl⁻ fluxmeasurements.

Ion flux experiments, pH stat studies, and trans-epithelial electricalmeasurements can elucidate the transport process in the non-IR and IRmice.

B) Determination of Functional Alterations in Nutrient Absorption due toIrradiation

Intestinal malabsorption of nutrients affects nutritional statusfollowing IR. As illustrated herein, selective absorption of nutrientsoccurs following IR. The presence of unabsorbed nutrients in the gutleads to osmotic diarrhea, which further complicates injury caused byirradiation. This Example illustrates the experimental design fordetermining the nutrients that are absorbed from the intestine after IR.

Easily-absorbed nutrients can be included into the therapeutic/dietarycomposition of the subject invention to examine the effect of various IRdoses on glucose absorption overtime.

Specifically, changes in glucose transport are determined in Ussingchambers following IR. The time required for glucose transport proteinsto return to their normal function (non-IR levels) is also investigated.The formulation (ORD) is derived according to the ability of the mice totolerate oral glucose. Glucose is withheld from the oral supportiveregimen until glucose transport begins to improve.

In addition, changes in amino acid (a.a) transport following IR areexamined. Electrogenic amino acid transport can be detected in an Ussingchamber as the net charge movement that occurs when the amino acid istransported. There is no charge movement associated with electroneutrala.a and, therefore, these transports are studied in brush bordermembrane vesicle studies (BBMV). Both electrogenic and electroneutrala.a are studied in BBMV for comparison between different experimentalmethods.

Specifically, the four major types of amino acid transport system arestudied by testing the uptake of representative amino acids L-leucine(neutral amino acid), L-proline (IMINO acid), L-glutamic acid (acidicamino acid), and L-cysteine (sulfurate amino acid) in brush-bordermembrane vesicles (BBMV) from non-IR and IR mice.

Changes in Electrogenic a.a Transport Due to IR

Amino acids are broadly classified into neutral, cationic and anionic astheir transport characteristics are largely based on charge (Table 4).Electrogenic a.a transport can occur via B^(0/+) (neutral and cationica.a) or X⁻ _(AG). Na-coupled and Na-independent a.a transport aredetermined by experiments in the presence and absence of lumen Na⁺. Inaddition, electroneutral a.a transport is studied in BBMV using ¹⁴Clabeled amino acids.

TABLE 4 Amino acid transport system in the brush-border membrane of thesmall intestine Depen- Involvement Transport Molecular Alternate denceof other system identity identity Substrates on Na ions B⁰ B⁰AT1 SLC6A19Neutral a.a Yes No B^(0/+) AT1B^(0/+) SLC6A14 Neutral a.a, Yes Cl⁻cationic a.a b^(0/+) b^(0/+)AT SLC7A9 Neutral a.a, No No cationic a.a,cystine rBAT SLC3A1 No transport function of its own, it influences thekinetic parameters of the transport function of b^(0/+)AT PAT PAT1SLC26A6 Neutral short No H⁺ chain a.a (glycine, alanine and proline)X_(AG) EAAT3 SLC1A1 Anionic a.a Yes K⁺, H⁺ (aspartate, glutamate)Preparation of BBMV to Study a.a Transport and Western Blot

BBMVs are isolated using the magnesium precipitation method⁷⁵. The totalprotein content of BBMVs is determined using the Bradford method⁷⁶.Vesicles are stored in liquid N₂ or at −80° C.

Assessment of Amino Acid Uptake by BBMVs

Amino acid uptake by BBMVs is performed at 25° C. using the rapidfiltration technique described by Hopfer et al.⁷⁵ with slightmodifications. BBMV suspensions (5 μl) are added to the incubationmedium (45 μl) containing 1 mmol/l of unlabeled amino acid, 25 μCi/ml ofradiolabeled substrate L-[U-¹⁴C]leucine, L-[U-14C]proline,L-[U-¹⁴C]glutamic acid, or L-[³⁵S]cysteine, 100 mmol/l NaSCN or KSCN,100 mmol/l mannitol, 0.1 mmol/l MgSO₄ and 10 mmol/l HEPES (pH 7.4). Thetime courses of the uptake of amino acids are measured in the presenceof Na⁺ gradient (using medium containing NaSCN) and in the absence ofNa⁺ gradient (medium containing KSCN). At specific time intervals, theuptake process is ended by adding 5 ml of ice-cold stop solutioncontaining 150 mmol/l KSCN and 10 mmol/l Tris-HEPES (pH 7.4). Thesuspension is immediately poured onto a pre-wetted Millipore filter thatis washed three times with 3 ml of ice-cold stop solution and immersedin 5 ml of scintillator Hisafe 3 fluid (LKB Products, Bromma, Sweden).The filter is then counted in a Liquid Scintillation Counter.Nonspecific binding to the filter is previously measured and subtractedfrom the total uptake. Results are expressed as picomoles of amino aciduptake per milligram of protein.

C) Determination of Changes in Paracellular Permeability Due to IR

Alterations in paracellular permeability are determined using thefollowing techniques. i) Dilution potential; ii) TEER; iii) permeationof large non ionic solutes of different sizes; FITC-conjugated dextranand Rhodamine B isothionate-Dextran.

Changes in Dilution Potential with Mitigation Following IR

Dilution potential measurements are used for determining the changes inthe permeability ratio between the Na⁺ and Cl⁻ using the Nernstequation. The results from these experiments are compared between non-IRand IR mice groups. The results from paracellular permeability andplasma endotoxin studies are correlated with the electrophysiology dataand survival data.

Dilution potentials are induced by mucosal perfusion with Ringersolutions containing various concentrations of Na⁺ and total osmolarityis adjusted with mannitol to maintain equal osmolarity betweenexperiments. The contribution of other ions to the conductance isestimated to be less than 5% and therefore is neglected. The potentialdifference across the membrane is measured using AgCl—AgCl electrodesand a multimeter (VCC MC8, Physiologic instruments Inc.). Dilutionpotentials are corrected for changes in junction potential (usually lessthan 1 mV). These experiments permit calculation of chloride and sodiumconductance of the paracellular pathway using the following formula.Em=RT/F*2.303 log 10{Pna [Na]_(D)+PCl[Cl]_(i)/ Pna [Na]_(l)+PCl[Cl]_(o)}R=8.314472 (J/K/mol); F=96.48531 (KJ/mol); Permeability ration (β)=PCl/PNa; T=310 (Kelvin)Changes in Non-Ionic Solute Permeation Through Paracellular SpacesFollowing IR

Paracellular permeability to water-soluble, uncharged solutes of varioussizes is studied in small intestine tissues mounted in an Ussing chamberusing FITC-conjugated dextran and Rhodamine B isothionate-Dextran(Sigma). These studies allow for the determination of paracellularpermeability changes due to IR.

Intercellular barrier formed by tight junctions is highly regulated andis size and ion-selective. Therefore, this intercellular barrierrepresents a semi-permeable diffusion barrier. Experiments are designedto determine the paracellular permeability to water-soluble, unchargedsolutes of various sizes in ileal or jejunal tissues mounted in Ussingchamber under basal conditions in both regular epithelium and epitheliumexposed to radiation.

FITC-conjugated dextran and Rhodamine B isothionate-Dextran (Sigma) at aconcentration of 3 mg/ml dissolved in Ringer solution is added to themucosal side of the Ussing chamber and maintained at 37° C. for 60 min.The solution in the basolateral bath solution is sampled to quantifyfluorescently labeled dextran. FITC-Dextran: Exc 485 nm and Em: 544 nmand Rhodamine B isothionate-Dextran: Exc 520 nm and Em 590 nm. Standardcurves are obtained from mice ileal or jejunal tissue mounted in Ussingchamber to check for any change in permeability with time. These valuesare then compared with tissues from IR- and non-IR mice.

D) Determination of Irradiation Effects

Tissues from mice sacrificed for an Ussing chamber and pH stat studiesare used for H&E staining, BrdU, stool formation, occult blood, bodyweight, immunohistochemistry and Western analysis. These results arethen compared to functional alterations in electrolyte, nutrient andparacellular permeability changes in non-IR and IR mice.

Pathological Analysis by Crypt Assay, H&E, BrdU Staining

a) Crypt Assay/Microcolony Survival Assay

Objective curves were fitted to the data, using a model for cellkilling, which assumes that clonogenic (‘structure-rescuing’) cells in amulticellular structure behave in accordance with Poisson statistics. Itis assumed that the structure remains intact until, on average, fewerthan three cells survive per structure; that survival of cells isexponential over the range of doses being analyzed; and that thestructure may regrow from one or more surviving cells. Each epithelialfocus is thought to represent survival of one or more clonogenic stemcell able to give rise to the regenerative crypt.

Mice are sacrificed at 3.5 days after IR for crypt microcolony assay.This interval is at or near the peak of mitotic recovery in crypts afterIR. It is used to study the acute effects of IR.

For the biological response to radiation, D₀ and D₁₀ values arecalculated. Studies have shown that despite lack of statisticallysignificant differences between the Do values, the variance about D₀greatly depended on the number of mice and sections per datum point.Decreased values of the coefficient of variance (˜5%) could not beobtained by increasing the number of sections above two and the numberof mice above four. Thus, the studies were designed with 3 sections permouse and six mice per datum point.

b) BrdU Staining to Detect Mitotic Activity after IR

The mice are injected with BrdU (30 mg/kg body weight) and animals aresacrificed at hours 12, 24, 48 or 72, when the tissues are alsoharvested for functional studies. BrdU injections are repeated onceevery 24 hrs, when BrdU labeling studies continue beyond 24 hrs aftertheir injection. After BrdU labeling, paraffin sections from mouse smallintestines are prepared and stained with anti-BrdU antibody (Ab). Cellswere scored per entire crypt and villous unit. At least 60 crypts andcorresponding villi were analyzed per mouse. BrdU-labeled cells werenormalized to total cell number per crypt or villous. The resultingpercentage is then plotted against the inductiontime. These studiesallow for the determination of the rate at which crypt progenitor cellstransit into the postmitoticvillous compartment, a direct correlation tothe rate of cell division in the crypt and kinetics of the migratingcrypt cells⁷⁷.

Changes in Physical Parameters with IR

Body weight, stool formation and fecal occult blood are studied in miceto detect the changes in the nutritional status of the animals with IR.For daily activity and signs of sickness, all of the mice are observedonce a day for diarrhea, lack of grooming, ruffled hair, decreasedeating and drinking habits, lethargy, etc, and recorded carefully.

Findings from these studies are compared to plasma analysis forsurrogate markers, pathological observations, Western blots,immunohistochemistry and functions studies.

Western Blot Analysis for Determining Molecular Alterations of TransportProcesses Involved in Electrolyte and Nutrient Transport

Changes in activities of the following transport proteins, which aredirectly or indirectly involved in electrolyte and nutrient absorption,are examined. The transport proteins include CFTR activity (correlatingwith electrogenic Cl⁻ secretion), NHE3 activity (correlates with Na⁺absorption), NBCe1-A/B activity in the villous (correlates with HCO₃ ⁻secretion), NKCC1 (basolateral uptake to Na⁺, K⁺ and Cl⁻ into the cell),SGLT-1 (glucose absorption), B⁰, B^(0/+), b^(0/+), PAT (proton-coupledelectrogenic transport system) and X⁻ _(AG) (Table 2). These studies arecompared to functional data in non-IR, IR and after treating with ORD.

Immunohistochemisty for Detection of Changes in the Expression Patternof Transport Proteins, Crypt and Villous Cell Markers

Frozen sections are made when the animals are sacrificed for functionalstudies and for immuno-staining using various antibodies that arespecific to various transporters (CFTR, NHE3, NKCC, NBCe1-A/B, SGLT, B⁰,B^(0/+), b^(0/+), PAT1, and X⁻ _(AG)). In addition, cell surface markerexpression patterns are examined to provide insights for crypt andvillous cell ratio. These studies allow for the determination ofalteration in the expression pattern of transporters with IR and ORDtreatment.

E) Identification of Surrogate Marker(s) for Radiation Effects

Although there are several studies trying to identify surrogatemarker(s) to determine the radiation dose and time since radiation fordetermining the onset of GI toxicity, these studies have been largelyunsuccessful. This Example illustrates experimental designs allowing forthe identification of surrogate marker(s) to predict the onset of GItoxicity, which may also prove useful in scenarios where multiple organsare involved.

Specifically, plasma is collected when the animals are sacrificed forfunctional evaluation (Ussing chambers). After exposure to an IR dose of0, 1, 3, 5, 7 or 9 Gy, the mice are sacrificed on day 1, 2, 3, 6, or 9.In order to identify surrogate markers, gut peptides, cytokines, andendotoxin are studied.

Plasma Analysis for Endotoxin

Plasma endotoxin levels are measured. Changes in plasma endotoxin levelsare correlated to changes in paracellular permeability, plasma gutpeptide levels, sickness and survival rate.

Plasma Analysis for Cytokines

Changes in plasma cytokine levels are examined using LUMINEX multiplexbead array technique in IR and non-IR mice.

Plasma Analysis for Gut Peptides

The gut-specific peptides, including insulin, glucagon, secretin,cholecystokinin, citrullin, somatostatin, peptide YY, ghrelin, NPY, andGLP-2, are investigated. All of the gut peptide kits were purchased fromPhoenix Pharmaceuticals, Inc. (CA, USA). Experiments are performedaccording to manufacture's instruction.

Statistical Analysis

Functional difference among the normal and IR tissues are compared. Thestatistical significance is calculated using the analysis of variance(ANOVA). The data are compared among the assays. The correlationcoefficient (R) is analyzed to determine the best functional marker. Allstatistical analyses are conducted using Version 9.1 of the SAS Systemfor Windows (Copyright© 2002-2003 SAS Institute Inc., Cary, N.C., USA.).If distributional assumptions associated with a particular statisticalprocedure are violated, appropriate transformations or non-parametricalternatives are used. Receiver Operating Characteristic (ROC) curvesare constructed and the areas under the ROC curves (AUCs) are comparedamong the various functional tests using the non-parametric method ofDeLong et al. (1988). The family-wise Type 1 error rate is controlled at0.05 using Tukey's method for multiple comparisons. The Pearsoncorrelation coefficients with associated p-value and 95% confidenceinterval are reported.

Example 16—Development of Ideal Oral Regimens for Treatment ofIR-Induced Gastrointestinal Injury

This Example illustrates experimental designs for developing oraltherapeutic compositions for treatment or amelioration ofradiation-induced Gl toxicity. It also determines the time when the oralrehydration diet (ORD) should start and how long the composition shouldbe administered after exposure to various doses of IR. The time forwhich ORD needs to be administered depends on the time needed for theK_(m) to return to the basal levels.

Methods

C57BL/6 mice (8 weeks old, male) from NCI are used. To determine theaffinity of the transporter, saturation kinetics is calculated by usingincreasing concentration of the respective nutrients. Preliminarystudies have shown that some a.a have increased absorption while someshowed decreased absorption, with changes in K_(m) and V_(max) after IR.Increasing concentration of the a.a are added to ileum or jejunum(subjected to separate evaluation) elicit an increase in I_(sc).Plotting known concentration of a.a against I_(sc) allows for thedetermination of the saturation kinetics. Administering the a.aselectively absorbed after IR via gastric lavage increases micesurvival. K_(m) and V_(max) for the nutrients are determined in mice IRwith 0, 1, 3, 5, 7 or 9 Gy on 0, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,25 and 30 days with 10 mice in each group.

A) Determination of K_(m) and V_(max) of Essential Amino Acids andGlucose for Development of Ideal Oral Radiation Diet (ORD)

As illustrated herein, irradiation causes changes in transport kineticsof nutrients, indicating altered affinity to respective transporters.The affinity for the glucose transporter determined using this techniqueshowed significant decrease and took approximately two weeks to returnto the base level. The presence of unabsorbed glucose or nutrients inthe gut lumen is known to cause diarrhea. K_(m) and V_(max) for thenutrients are determined in mice exposed to different doses of IR andfollowed up for a period up to 30 day after IR. These studies are usefulfor formulating an ORD based on their absorption pattern with time andradiation dose. In addition, the nutrients that show increasedabsorption after IR may be utilized as alternate sources of energy forthe system. The formulation (ORD) will then be used in survival studies.

Changes in K_(m) and V_(max) for glucose transport in Ussing chambersfollowing IR

Glucose transport is studied. Specifically, K_(m) and V_(max) forglucose are studied. Increasing concentrations of glucose are added tothe lumen side in Ussing chamber experiments and increase in I_(sc)recorded. Glucose is withheld from the oral supportive regimen untilglucose transport begins to improve. The formulation is based on theability of the mice to tolerate oral glucose.

Changes in K_(m) and V_(max) for Amino Acid (a.a) Transport Following IR

The kinetic pattern of amino acids based on the IR dose and timefollowing IR is studied by determining the K_(m) and V_(max) for eacha.a. Kinetic indices of electrogenic a.a are determined in Ussingchamber setting as described. Briefly, increasing the concentration ofthe a.a added to the lumen solution results in increasing I_(sc)response, with saturation at particular a.a concentration. K_(m) andV_(max) are calculated from the saturation curve.

Electroneutral a.a are studied using BBMV. Amino acid uptake by BBMV isperformed in the presence of different concentrations of substrate, from0.025 to 7 mmol/l, at a fixed transport time of 3 s (19). Each assay isperformed in triplicate using the pool of BBMV (n=12) from eachexperimental group. Maximal velocity (V_(max)) is expressed as picomolesof substrate per milligram of protein in 3 s, and the transporteraffinity constant (K_(m)) is expressed as millimoles per liter.

Optimize the ORD therapy to mitigate GI toxicity and improve survival

It is discovered that lysine at a dose range of 20 mg/mice/day canincrease survival in mice. To optimize the ORD treatment regimen byselecting a proper administration dose, frequency and interval, theanalysis of the effects of ORD on survival at 7 days, stool formation,occult blood and body weight is performed. ORD is initiated as early as3 hours after a lethal dose of IR (15.6 Gy=1.2×LD_(50/7) value) at adose range determined from K_(m) values of respective a.a or glucose.The concentration of glucose or a.a used for gastric lavage iscalculated from K_(m) based on recommended daily amounts currently inuse for glucose and essential amino acids in adult humans. The dosetranslation from human to mice is based on the K_(m) factors⁷⁸. Thus, aninverse relationship exists between the K_(m) and the daily dose for thenutrients. If IR increased the K_(m) (suggesting decreased affinity forthe transporter), then there is a proportionate decrease in daily dosefor the respective nutrient. Two additional ORDs are formulated withdoses, i.e., 3 times above and below the calculated dose. The best ORDdosage is determined based on the survival studies.

The gastric lavage is repeated once daily for 7 days. The dose frequencyand interval of ORD gastric lavage are subject to change according tothe results of the survival studies. GI toxicity peaks around day 2-3after IR and then gradually recovers by 7 days if the ORD is effective.The mice are observed daily up to 7 days after IR to monitor theirsurvival.

All the mice receiving regular diet die or are sacrificed (moribund;defined as a combination of 20% weight loss, failure to groom, reducedactivity and decreased inquisitiveness) within 7 days after IR. If themice receiving ORD treatment are protected from IR-induced lethality,then the survival experiment will be repeated with an additional 10mice/group with same treatment to ensure the results are reproducible.The survival data will be analyzed by the Fisher's exact test.

The sample size of 10 animals per group ensures sufficiently high power(>80%) to detect survival differences between close to 0% for thevehicle group and 60% or higher for each of the intervention (in apair-wise comparison carried out at the adjusted alpha level of0.017≈0.05/3) to ensure an overall alpha level of 5%. In the event thata statistically significant difference is not observed or only partialmitigation is achieved by the ORD treatment, a new cycle of regimenoptimization will be undertaken as described earlier to ensure maximalmitigating efficacy against IR-induced lethality. After selection of anoptimal dose, whether more frequent (twice daily) ORD gastric lavage isrequired is evaluated to achieve greater radiation mitigation and morerapid crypt recovery.

Determining the DMF and the Window of Effectiveness of ORD for Post-IRTherapy

Dose modification factor (DMF) is one of the most important parametersto measure the effectiveness of a radiation mitigator, which is definedby DMF=LD₅₀ ^(T)/LD₅₀ ^(C) where T is ORD treatment group and C denotesthe control group on regular diet⁵¹. To determine the efficacy of ORDtreatment in mitigating IR-induced lethality, groups of average 10C57BL/6 mice (10-20 mice/group varying with IR dose) are treated withvehicle or ORD using the optimal regimen defined by the previousexperiments. The vehicle-treated mice are exposed to 11 Gy to 13 Gy IRusing 0.5 to 1 Gy increments. The survival of these mice is recordedduring a 7-day observation period after IR. Mice are euthanized at theend of the observation period or when they become moribund.

Small intestine and plasma are collected after euthanasia. Blood samplesare used for gut peptide analysis, while the small intestine tissuespecimens are used for investigating IR-induced intestinal damage. TheLD_(50/7) value is a good indicator of IR-induced GI toxicity.

LD_(50/7) for the vehicle-treated mice is close to 13 Gy, based onprevious observation in our laboratory. The ORD treated groups areexposed to IR ranging from 14.5 to 16.5 Gy IR with 0.5 to 1 Gyincrements, observed and examined as described above for vehicle-treatedmice. If substantial numbers of mice in ORD treatment groups surviveeven after exposed to 16.5 Gy, higher IR doses are given to mice in asubsequent study. The LD_(50/7) value is calculated for ORD-treatedanimals based on their survival curves and then, the DMF for ORD iscalculated. ORD-treated mice have a DMF for LD_(50/7) greater than 1.2.

To determine how soon the ORD treatment should be given after IR, fivegroups of animals are administered with ORD at 0, 1, 3, 5, 7, 9, 12 and24 hours post-IR and followed up with scheduled ORD treatment andobserved for 7 days, along with a positive control (3 h post-IRtreatment) and a negative control (saline vehicle). Survival of theanimals is compared based on survival at the 7-day time point.

In this model, a number of logistic-regression models (outcome variabledead/alive at 7 days) and various time trends in the eight groups havingadministered ORD after IR are considered. Both linear (most likelydecreasing survival as treatment delay increases) and non-linear(exponential survival decreases) models are considered.

Comparisons versus 3 h post-IR administration and vehicle are performedin a pair-wise fashion using the Fisher's exact test. There is apair-wise comparison (different timing groups vs. the 3 h post-IR, ORDgroup and vs. vehicle) and the individual test alpha level will bemaintained at 0.005 (≈0.05/10).

a) Survival Rate:

A major index for the treatment effect of ORD is to determine survivalrate. It is recorded twice a day and a survival curve created.

b) Daily Activity or Signs of Sickness

All of the mice are observed once a day for the signs of sickness, suchas diarrhea, lack of grooming, ruffled hair, decreased eating anddrinking habits, lethargy, etc, and recorded carefully.

c) Body Weight, Stool Formation and Occult Blood

To determine if the ORD could reverse some of the effects fromIR-induced GI toxicity, the colon will be removed and pictured for stoolformation and feces analyzed for occult blood, when those animals aresacrificed for functional studies as described herein. These studiesallows for determining if the mitigation agents are able to maintain theintegrity of GI mucosa and their function that are visible to the nakedeye.

d) Immunohistochemistry

Inflammatory cell infiltration in the lamina propria is analyzed usingH&E stained sections from jejunum or ileum. Care will be taken todetermine the distribution frequency of lymphoid follicles.

The optimal dose, starting time and schedule of ORD for acute GItoxicity are determined in a sequence. Mice are treated with differentdose formulation of ORD after IR exposure. The optimal dose isdetermined in logistic regression models by determining survival over 7days (yes versus no) as the response variable and dose level as theexplanatory variable. Due to the uncertainty of the dose-response curve,several plausible dose-response models are proposed. After the model ofdose-response is determined, the minimum effective dose (MED) iscalculated. Starting Time and optimal duration of the therapy areanswered by equivalence tests using the estimated mean responses andvariance in the ANOVA model.

Example 17—Determination of Functional Improvement in GI Function

In this Example, electrophysiology experiments are performed todetermine how ORD helps restore IR-injured gut mucosa to absorbelectrolytes and nutrients. Functional changes are correlated to plasmasurrogate marker(s), cytology, and physical observations such as dailyactivity, body weight, and stool formation. Fecal occult blood, cytologysuch as crypt assay, H&E staining, BrdU staining, immunohistochemistryand Western blot analysis. These studies allow for the determination ofthe protective effects of ORD on GI function at molecular, cellular andfunctional level.

Methods

C57BL/6 mice (8 weeks old, male) from NCI are used. Functional studies,physical observations, cytology, immunohistochemistry, Western analysisare performed and plasma surrogate markers are used as specific indicesfor IR-induced GI toxicity. Mice were randomly divided into groups andthe abdomen irradiated with a Shepherd Mark-I using a Cs sourcedelivering IR at 1.84 Gy/min dose rate. Mice are irradiated with 1, 3,5, 7 or 9 Gy and then are administered ORD. Mice are treated with ORD.Mice are sacrificed on day 6 and tissues are used for functional,histopathology, Western blot and immunohistochemistry.

A) Correlation of Effects of ORD with Functional Improvement inElectrolyte and Nutrient Absorption

A set of indices are used to evaluate the treatment effect: 1) the miceare weighted daily and closely observed for any sickness signs; 2) bloodsamples and physical parameters are analyzed when the animals aresacrificed for functional studies (electrolyte and nutrient absorption),crypt assay, immunohistochemistry and western blot analysis. Bloodsamples are used for measuring plasma endotoxin (an index for gutbarrier dysfunction), cytokines, gut peptides (insulin, Glucagon,secretin, cholecystokinin, citrullin, somatostatin, peptide YY, Ghrelin,NPY and GLP2), citrulline, glucose and insulin.

Determination of Transepithelial Flux of Na⁺ and Cl⁻ in Ussing ChamberStudies

To investigate functional improvement of ORD, jejunum and ileum sheets(subjected to separate evaluation) obtained from mice are mounted inUssing chamber and experiments are performed as described in Example 15.Na⁺ and Cl⁻ absorption are compared between non-IR, IR and ORD treatedmice groups.

Determination Pf HCO₃ ⁻ Secretion Using pH Stat Techniques

Experiments are performed as described in Example 15. Restoration ofHCO₃ ⁻ secretion with ORD treatment suggests functional improvement.HCO₃ ⁻ secretion is compared between non-IR, IR and ORD treated micegroups.

Determination of Nutrient Absorption in Ussing Chamber and VesicleStudies

As described in Example 15, glucose, electrogenic a.a and electroneutrala.a absorption is determined. Results from these studies are comparedbetween non-IR, IR and ORD treated mice groups.

Determination of Changes in Paracellular Permeability with MitigationFollowing IR

A decrease in paracellular permeability with ORD treatment suggestsimprovement in epithelial integrity. These changes will indicateconcomitant improvement in plasma endotoxin level.

Correlate Effects of ORD with Crypt Assay, H&E Staining, BrdU, StoolFormation, Occult Blood, Body Weight, Immunohistochemistry and WesternAnalysis

The studies will be similar to that described earlier in Example 15 andthe results will be compared between non-IR, IR and ORD treated micegroups.

Histopathological Analysis to Determine Anatomical Improvement

Specimens will be processed for H&E staining and pathological analysis,including the crypt assay, BrdU staining as described in Example 15.Briefly, the tissues will be fixed in formalin, processed in paraffinblocks and stained with H & E.

Immunohistochemisty to Detect Changes in the Expression Pattern ofTransport Proteins, Crypt and Villous Cell Marker

The tissues harvested will be used for immuno-statining using variousantibodies that are specific to various transporters (NHE3, NBCe1-A/B,SGLT, B^(0/+), b^(0/+), X⁻ _(AG)) and cell surface markers (Lgr5, EphB2and EphB3). The method will be similar to that described in Example 15.These studies will help determine the extent of villous and crypt cellformation following treatment with ORD.

Western Blot Analysis to Study Molecular Alterations of TransportProcesses Involved in Electrolyte and Nutrient Transport

The method will be similar to that described in Example 15. CFTRactivity (correlating with electrogenic Cl⁻ secretion), NHE3 activity(correlates with Na⁺ absorption), NBCe1-A/B activity in the villous(correlates with HCO₃ ⁻ secretion), SGLT-1, B^(0/+), b^(0/+) or X⁻ _(AG)will be examined.

Correlate Effects of ORD Using Plasma Analysis of Surrogate Marker(s)

Preliminary studies have shown changes in gut peptides following IR inmice. Changes in surrogate marker levels toward basal levels areexamined and the results will indicate systemic improvement with ORDtreatment.

Statistical Analysis

The mean and standard deviation of the raw data are calculated andgraphical techniques such as bar chart will be applied. The mainapproach to comparing these two groups (treatment vs. vehicle) utilizesmixed effect models (linear or non-linear) based on longitudinal data.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

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We claim:
 1. A method for promoting intestinal health in a subject inneed thereof, the method comprising: administering a formulationcomprising free amino acids to the subject in need thereof, wherein thefree amino acids comprise, as free amino acids, a therapeuticallyeffective amount of serine and valine; and a therapeutically effectiveamount of at least one additional free amino acid of threonine, lysine,glycine, aspartic acid, isoleucine, tryptophan, or tyrosine, or anycombination thereof; wherein the therapeutically effective amount of theserine and valine and the therapeutically effective amount of the atleast one additional free amino acid promotes intestinal health in thesubject; wherein the formulation does not comprise glucose, or when theformulation does comprise glucose, the glucose is present in aconcentration equal to or less than 10 mM; wherein the formulation doesnot comprise: free amino acid glutamine; a glutamine-containingdipeptide; or any combination thereof; wherein the formulation does notcomprise riboflavin; and wherein a therapeutically effective amount ofthe formulation promotes intestinal health in the subject.
 2. The methodof claim 1, wherein the subject has an injury to small intestinalepithelial cells.
 3. The method of claim 2, wherein the injury to smallintestinal epithelial cells comprises injury caused by disease,radiation, chemotherapy, proton therapy, abdominal surgery, and/or atleast one cytotoxic agent.
 4. The method of claim 3, wherein the diseasecomprises inflammatory bowel disease (IBD), ulcerative colitis, duodenalulcers, or Crohn's disease.
 5. The method of claim 1, wherein thesubject is a human.
 6. The method of claim 1, wherein the free aminoacids consist essentially of: serine; valine; and at least one of:threonine, lysine, glycine, aspartic acid, isoleucine, tryptophan,tyrosine, or any combination thereof.
 7. The method of claim 1, whereinthe free amino acids consist of: serine; valine; and at least one of:threonine, lysine, glycine, aspartic acid, isoleucine, tryptophan,tyrosine, or any combination thereof.
 8. The method of claim 1, whereinthe formulation further comprises water.
 9. The method of claim 1,wherein the formulation further comprises electrolytes, vitamins, and/orminerals, wherein the vitamins do not comprise riboflavin.
 10. Themethod of claim 1, wherein the glucose concentration is less than 4 mM.11. The method of claim 1, wherein the formulation is sterile.
 12. Themethod of claim 1, wherein the formulation comprises lysine, glycine,aspartic acid, and/or isoleucine.
 13. The method of claim 1, wherein theformulation comprises tyrosine, aspartic acid, threonine, valine, andserine.
 14. The method of claim 1, wherein the free amino acids consistessentially of tyrosine, aspartic acid, threonine, valine, and serine.15. The method of claim 1, wherein the free amino acids consist oftyrosine, aspartic acid, threonine, valine, and serine.
 16. The methodof claim 1, wherein serine is present at a concentration of about 420 to3784 mg/l and valine is present at a concentration of about 469 to 4217mg/l.
 17. The method of claim 1, wherein threonine if present is at aconcentration of about 476 to 4288 mg/l; lysine if present is at aconcentration of about 730 to 6575 mg/l; glycine if present is at aconcentration of about 300 to 2703 mg/l; aspartic acid if present is ata concentration of about 532 to 4792 mg/l; isoleucine if present is at aconcentration of about 525 to 4722 mg/l; and tryptophan if present is ata concentration of about 817 to 7352 mg/l.
 18. The method of claim 1,wherein the free amino acids consist essentially of: serine; valine;threonine, lysine, glycine, aspartic acid, isoleucine, tyrosine, andoptionally, tryptophan.
 19. The method of claim 3, wherein thechemotherapy or the at least one cytotoxic agent comprises treatmentwith cisplatin, 5-fluorouracil (5-FU), hydroxyurea, etoposide,arabinoside, 6-mercaptopurine, 6-thioguanine, fludarabine, methothexate,or steroids, or a combination thereof.
 20. The method of claim 3,wherein the formulation is administered for a period of 1 to 14 daysafter the subject receives the radiation, the chemotherapy, the protontherapy, or the at least one cytotoxic agent.
 21. The method of claim 3,wherein the subject has radiation enteritis.